U.S. patent number 7,407,780 [Application Number 11/598,375] was granted by the patent office on 2008-08-05 for process for producing glycerol in recombinant bacterial host cells.
This patent grant is currently assigned to Genencor International, Inc.. Invention is credited to Timothy C. Dodge, Fernando Valle.
United States Patent |
7,407,780 |
Dodge , et al. |
August 5, 2008 |
Process for producing glycerol in recombinant bacterial host
cells
Abstract
The invention provides methods for producing products comprising
improved host cells genetically engineered to have uncoupled
productive and catabolic pathways. In particular, the present
invention provides host cells having a modification in nucleic acid
encoding an endogenous enzymatic activity that phosphorylates
D-glucose at its 6th carbon and/or a modification of nucleic acid
encoding an enzymatic activity that phosphorylates D-gluconate at
its 6th carbon. Such improved host cells are used for the
production of products, such as, ascorbic acid intermediates.
Methods for making and using the improved host cells are provided.
Nucleic acid and amino acid sequences for glucokinase and
gluconokinase are provided.
Inventors: |
Dodge; Timothy C. (Sunnyvale,
CA), Valle; Fernando (Burlingame, CA) |
Assignee: |
Genencor International, Inc.
(Palo Alto, CA)
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Family
ID: |
26960977 |
Appl.
No.: |
11/598,375 |
Filed: |
November 13, 2006 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20070059811 A1 |
Mar 15, 2007 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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10117283 |
Apr 4, 2002 |
7241587 |
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Current U.S.
Class: |
435/126; 435/183;
435/194; 435/252.3; 435/471; 536/23.2 |
Current CPC
Class: |
C12N
9/1205 (20130101); C12N 15/52 (20130101); C12P
25/00 (20130101); C12P 19/02 (20130101); C12P
7/60 (20130101) |
Current International
Class: |
C12P
17/04 (20060101); C07H 21/04 (20060101); C12N
1/20 (20060101); C12N 15/74 (20060101); C12N
9/12 (20060101) |
Field of
Search: |
;435/126,183,194,252.3,471 ;536/23.2 |
References Cited
[Referenced By]
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EP |
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Oct 1992 |
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WO |
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WO 98/21340 |
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May 1998 |
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WO |
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WO |
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Jun 1999 |
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WO |
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WO |
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WO 99/61623 |
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Dec 1999 |
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WO |
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WO 00/37667 |
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Jun 2000 |
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WO |
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WO 01/12833 |
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Feb 2001 |
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WO |
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|
Primary Examiner: Saidha; Tekchand
Assistant Examiner: Fronda; Christian L.
Attorney, Agent or Firm: Marcus-Wyner; Lynn
Government Interests
STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSORED
RESEARCH
This invention was made with the United States Government support
under Award No. 70 NANB 5H1138 awarded by the United States
Department of Commerce. The Government has certain rights in this
invention.
Parent Case Text
This is a continuation of application Ser. No. 10/117,283 filed
Apr. 4, 2002 now U.S. Pat. No. 7,241,587.
Claims
We claim:
1. A process for producing glycerol in a recombinant bacterial host
cell comprising, culturing a bacterial host cell in the presence of
a carbon source under conditions suitable for the production of
glycerol, wherein an endogenous polynucleotide of the bacterial
host cell which encodes a glycerol kinase comprising at least 95%
sequence identity to SEQ ID NO:14 has been inactivated and allowing
the production of glycerol from the carbon source in said
recombinant bacterial host cell.
2. The process according to claim 1, wherein the glycerol kinase
comprises at least 99% sequence identity to SEQ ID NO:14.
3. The process according to claim 2, wherein the glycerol kinase
comprises the amino acid sequence of SEQ ID NO:14.
4. The process according to claim 1, wherein the polynucleotide
comprises the sequence of SEQ ID NO:13.
5. The process according to claim 1, wherein inactivation is by
deletion of the polynucleotide encoding said glycerol kinase.
6. The process according to claim 1, wherein the bacterial host is
selected from the group consisting of Erwinia, Enterobacter,
Corynebacreria, Acetobacter, Gluconobacter, Pantoea, Pseudomonas,
Bacillus, and Escherichia cells.
7. The process according to claim 6, wherein the bacterial host is
selected from the group consisting of Erwinia, Enterobacter, and
Pantoea cells.
8. The process according to claim 7, wherein the bacterial host is
a Pantoea cell.
9. The process according to claim 1 further comprising isolating
the glycerol.
10. The process according to claim 1, wherein the recombinant
bacterial cells are cultured by continuous cell culture.
11. The process according to claim 1, wherein the recombinant
bacterial cells are cultured by batch culture.
12. The process according to claim 6, wherein the bacterial host is
cultured in the presence of a carbon source comprising
fructose.
13. The process according to claim 8, wherein the Pantoea is
cultured in the presence of a carbon source comprising
fructose.
14. The process according to claim 8, wherein the Pantoea is P.
citrea.
Description
TECHNICAL FIELD
The present invention relates to engineering of metabolic pathways
of host cells and provides methods and systems for the production
of products in host cells. In particular, the invention provides
methods for producing products in host cells which have been
genetically engineered to have uncoupled productive and catabolic
pathways.
BACKGROUND ART
In the initial stage of host cell carbohydrate metabolism, that is,
glycolysis, each glucose molecule is converted to two molecules of
pyruvate in the cytosol. The chemical reactions that convert
glucose to pyruvate are referred to as the Embden-Meyerhoff
pathway. All of the metabolic intermediates between the initial
carbohydrate and the final product, pyruvate, are phosphorylated
compounds. The final stage of oxidation of carbohydrates, the
citric acid cycle, is a complex set of reactions that also takes
place in the cytosol. The reactions in the Embden-Meyerhoff pathway
and citric acid cycle result in the conversion of carbohydrate
molecules to CO.sub.2 molecules with the concomitant reduction of
NAD+ to NADH molecules and the formation of ATP.
The central metabolic routes produce NADH or NADPH. In general
NADPH is utilized in biosynthetic reactions and NADH is rapidly
reoxidized in two ways: (1) In fermentative pathways by the direct
reduction of organic metabolites. (2) In respiratory processes by
electron transport through a respiratory chain to a terminal
electron acceptor. This acceptor is usually O.sub.2, but in some
cases can be productive ions, including nitrate and sulfate. In all
respiratory processes, ATP is generated. Some bacteria posses the
ability to oxidize some substrates extracellularly, producing
useful oxidation products such as L-sorbose, D-gluconate,
keto-gluconates, etc. Such oxidation reactions are called
productive fermentation since they involve incomplete substrate
oxidation, accompanying accumulation of corresponding oxidation
product in large amounts in the growth medium. The oxidation
reaction is coupled to the respiratory chain of the microorganism.
(Bacterial Metabolism 2.sup.nd Edition (1985) Springer-Verlag, New
York, N.Y.).
Bacteria which ferment glucose through the Embden-Meyerhof pathway,
such as members of Enterobacteriacea and Vibrionaceae, are
described in Bouvet, et al. (1989) International Journal of
Systematic Bacteriology, 39:61-67. Pathways for metabolism of
ketoaldonic acids in Erwinia sp. are described in Truesdell, et al,
(1991) Journal of Bacteriology, 173:6651-6656.
Host cells having mutations in enzymes involved in glycolysis have
been described. Yeast having mutations in glucokinase are described
in Harrod, et al. (1997) J. Ind. Microbiol. Biotechnol. 18:379-383;
Wedlock, et al., (1989) J. Gen. Microbiol. 135: 2013-2018; and
Walsh et al. (1983) J. Bacteriol. 154:1002-1004. Bacteria deficient
in glucokinase have been described. Pediococcus sp. deficient in
glucokinase is described in Japanese patent publication JP 4267860.
Bacillus sphaericus lacking glucokinase is described in Russell et
al. (1989) Appl. Environ. Microbiol. 55: 294-297. Penicillium
chrysogenum deficient in glucokinase is described in Barredo et al.
(1988) Antimicrob. Agents-Chemother 32:1061-1067. A
glucokinase-deficient mutant of Zymomonas mobilis is described in
DiMarco et al. (1985) Appl. Environ. Microbiol. 49:151-157.
Many bacteria posses an active transport system known as
Phosphotransferase transport System (PTS) that couples the
transport of a carbon source to its phosphorylation. In this
system, the phosphoryl group is transferred sequentially from
phosphoenolpyruvate (PEP) to enzyme I and from enzyme I to protein
HPr. The actual translocation step is catalyzed by a family of
membrane bound enzymes (called enzyme II), each of which is
specific for one or a few carbon sources. Considering that PTS
consumes PEP to phosphorylate the carbon source, and PEP is a
central metabolite used in for many biosynthetic reactions, it may
decrease the efficiency of conversion of a carbon source into a
desired product. this transport system has been replaced by a
permease and glucokinase from an heterologous origin as described
by Parker et al. (1995) Mol. Microbiol. 15: 795-802. or homologous
origin as reported by Flores et al. (1996) Nat. Biotechnol. 14:
620-623. In both of these 2 examples, the function of the PTS
system for glucose transport and phosphorylation was replaced by a
glucose permease and a glucokinase activities.
Products of commercial interest that have been produced
biocatalytically in genetically engineered host cells include
intermediates of L-ascorbic acid; 1,3-propanediol; glycerol;
D-gluconic acid; aromatic amino acids;
3-deozy-D-arabino-heptulosonate 7-phosphate (DAHP); and catechol,
among others.
L-Ascorbic acid (vitamin C, ASA) finds use in the pharmaceutical
and food industry as a vitamin and antioxidant. The synthesis of
ASA has received considerable attention over many years due to its
relatively large market volume and high value as a specialty
chemical.
The Reichstein-Grussner method, a chemical synthesis route from
glucose to ASA, was first disclosed in 1934 (Helv. Chim. Acta
17:311-328). Lazarus et al. (1989, "Vitamin C: Bioconversion via a
Recombinant DNA Approach", Genetics and Molecular Biology of
Industrial Microorganisms, American Society for Microbiology,
Washington D.C. Edited by C. L. Hershberger) disclose a
bioconversion method for production of an intermediate of ASA,
2-keto-L-gulonic acid (2-KLG, KLG) which can be chemically
converted to ASA. This bioconversion of carbon source to KLG
involves a variety of intermediates, the enzymatic process being
associated with co-factor dependent 2,5-DKG reductase activity
(2,5-DKGR or DKGR).
Many bacterial species have been found to contain DKGR,
particularly members of the Coryneform group, including the genera
Corynebacterium, Brevibacterium, and Arthrobacter. DKGR obtained
from Corynebacterium sp. strain SHS752001 is described in Grindley
et al. (1988, Applied and Environmental Microbiology 54:
1770-1775). DKGR from Erwinia herbicola is disclosed in U.S. Pat.
No. 5,008,193 to Anderson et al. Other reductases are disclosed in
U.S. Pat. Nos. 5,795,761; 5,376,544; 5,583,025; 4,757,012;
4,758,514; 5,004,690; and 5,032,514.
1,3-Propanediol is an intermediate in the production of polyester
fibers and the manufacture of polyurethanes and cyclic compounds.
The production of 1,3-propanediol is described in U.S. Pat. Nos.
6,025,184 and 5,686,286. 1,3-propanediol can be produced by the
fermentation of glycerol. The production of glycerol is described
in WO 99/28480 and WO 98/21340.
D-gluconic acid and its derivatives have been used commercially as
agents in textile bleaching and detergents. The production of
D-gluconic acid in Bacillus species lacking gluconokinase activity
and having high glucose dehydrogenase activity is described in WO
92/18637.
The production of members of the aspartate family of amino acids is
described in U.S. Pat. No. 5,939,307. The production of riboflavin
(Vitamin B2) is described in WO 99/61623.
Many cyclic and aromatic metabolites are derived from DHAP
including tyrosine, tryptophan and phenylalanine. The production of
DAHP is described in U.S. Pat. No. 5,985,617. Catechol is a
starting material for the synthesis of pharmaceuticals, pesticides,
flavors, fragrances and polymerization inhibitors. The production
of catechol is described in U.S. Pat. No. 5,272,073.
However, there are still problems associated with these production
methodologies. One such problem is the diversion of carbon
substrates from the desired productive pathways to the catabolic
pathways. Such diversion results in the loss of available carbon
substrate material for conversion to the desired productive pathway
products and resultant energy costs, ATP or NADPH, associated with
the transport or phosphorylation of the substrate for catabolic
pathway use.
In spite of the advances made in the production of products by host
cells, there remains a need for improved host cells for use in the
production of desired products. The present invention addresses
that need.
SUMMARY
Methods for the production of products in recombinant host cells
genetically engineered to have uncoupled productive and catabolic
pathways during part or all of the production are provided. The
present invention also provides recombinant host cells genetically
engineered to comprise productive and/or catabolic pathways that
are uncoupled or that can be regulated during production, and
methods for their preparation.
Accordingly, the invention provides a process for producing a
product in a recombinant host cell comprising, culturing a host
cell capable of producing said product in the presence of a carbon
source under conditions suitable for the production of said product
wherein said host cell comprises productive and catabolic pathways,
wherein said pathways are uncoupled during part or all of said
culturing. In some embodiments, the productive pathway and
catabolic pathway are uncoupled during all of said culturing. In
some embodiments, the product being produced is a component of the
productive pathway or the host cell. In other embodiments, the
product being produced is a component of the catabolic pathway of
the host cell. In further embodiments, the product being produced
is encoded by nucleic acid recombinantly introduced into the host
cell.
In some embodiments, the productive pathway is in the host cell
membrane. In other embodiments, the catabolic pathway is
intracellular. In further embodiments, the productive pathway and
catabolic pathway are uncoupled at the stage of initial
phosphorylation of said carbon source. In additional embodiments,
the productive pathway and catabolic pathway are uncoupled at the
stage of phosphorylation of a carbon metabolite.
In further embodiments, the uncoupling of the productive pathway
and catabolic pathway comprises inhibition of at least one
enzymatic activity that phosphorylates a carbon source and/or a
carbon metabolite during said culturing. In other embodiments, the
uncoupling of said productive pathway and said catabolic pathway
comprises inactivation of at least one enzymatic activity that
phosphorylates said carbon source and/or a carbon metabolite during
part or all of said culturing.
In additional embodiments, the host cell comprises a mutation in or
deletion of part or all of a polynucleotide that encodes an
enzymatic activity that couples an productive pathway with a
catabolic pathway. In yet other embodiments, the host cell
comprises at least one polynucleotide that lacks the encoding for
an enzymatic activity that phosphorylates said carbon source and/or
a carbon metabolite wherein said polynucleotide is operably linked
to a regulatable promoter.
In some embodiments, the enzymatic activities that are reduced or
inactivated are those that phosphorylate D-glucose at its 6th
position. In other embodiments, the enzymatic activity that is
reduced or inactivated phosphorylates D-gluconic acid at its 6th
position. In further embodiments, the enzymatic activity that
phosphorylates D-glucose at its 6th carbon includes glucokinase,
phosphoenol pyruvate synthase (PEP) or phosphotransferase system
(PTS). In additional embodiments, the enzymatic activity that
phosphorylates D-gluconate at its 6th carbon includes
gluconokinase.
In some embodiments, the product is recovering and in other
embodiments, the product is converted into a second product. The
host cell includes Gram negative or Gram positive host cells. In
some embodiments, the host cell is an Enterobacteriaceae host cell
that includes Erwinia, Enterobacter, Gluconobacter, Acetobacter,
Coymebacteria, Escherichia or Pantoea. In other embodiments, the
host cell is an that includes Bacillus and Pseudomonas.
In other embodiments, the host cell can be any bacteria that
naturally or after proper genetic modifications, is able to utilize
one carbon source to maintain certain cell functions, for example,
but not limited to, the generation of reducing power in the form of
NAD, FADH.sub.2 or NADPH, while another carbon source is converted
into one or more product(s) of commercial interest.
In some embodiments, the uncoupling of the productive and catabolic
pathways allows the production of compounds generally derived from
the catabolic pathway, wherein those products generally derived
from the productive pathways are utilized to satisfy the metabolic
demands of the host cell. In other embodiments, the uncoupling of
the productive and catabolic pathway allows the production of
compounds generally derived from the productive pathway, whereas
those products derived from compounds present in the catabolic
pathway satisfy the metabolic demands of the host cell.
In some embodiments, the product includes those products generally
derived from the catabolic pathway include those derived from
fructose, the pentose pathway and the TCA cycle. In other
embodiments, the product includes those generally derived from the
productive pathway, e.g., an ascorbic acid intermediate including
GA, KDG, DKG, KLG or IA.
The invention also provides host cells comprising an productive
pathway and a catabolic pathway, wherein said productive pathway
and said catabolic pathways are uncoupled. In some embodiments, the
host cells comprise a modification of the polynucleotide encoding
an enzymatic activity such that such enzymatic activity is reduced
or inactivated. One such modification precludes the host cell from
phosphorylating D-glucose at it 6th carbon and/or precludes a host
cell from phosphorylating D-gluconic acid at its 6th carbon,
wherein one or both of said polynucleotides is modified. In some
embodiments, the enzymatic pathway that is inactivated includes
that of hexokinase, glucokinase; gluconokinase; phosphoenol
pyruvate synthase (PEP); or phosphotransferase system (PTS).
The present invention also provides methods for producing host
cells having modified levels of enzymatic activities. The present
invention also provides novel nucleic acid and amino acid sequences
for which lack enzymatic activity that phosphorylates D-glucose at
its 6th carbon and enzymatic activity that phosphorylates
D-gluconate at its 6th carbon.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides a schematic representation of some of the metabolic
routes involved in Glucose assimilation in Pantoea citrea. The
enzymatic steps affected by the genetic modifications described in
the present invention, are indicated by an X. Boxes labeled with a
T represent putative transporters.
FIG. 2. Some possible catabolic routes that can be used to channel
glucose into cellular metabolism. The arrows represent at least one
enzymatic step.
FIG. 3. depicts products that can be obtained from indicated
commercial routes. The majority of the carbon used to synthesize
the compounds listed on the left side, can be obtained from the
catabolic pathway or TCA cycle. On the contrary, the compounds on
the right, derive most of its carbon from the pentose pathway
and/or from the oxidation of glucose into keto-acids.
FIG. 4 depicts a nucleic acid (SEQ ID NO:1) for a Pantoea citrea
glucokinase
FIG. 5 depicts an amino acid (SEQ ID NO:2) sequence for a Pantoea
citrea glucokinase.
FIG. 6 depicts a nucleic acid (SEQ ID NO:3) for a Pantoea citrea
gluconokinase
FIG. 7 depicts an amino acid (SEQ ID NO:4) sequence for a Pantoea
citrea gluconokinase.
FIG. 8 depicts amino acid (SEQ ID NO: 8-13) for the genes glk 30,
glk 31, gnt 1, gnt 2, pcgnt 3 and pcgnt 4.
FIG. 9 depicts D-glucose, D-gluconate and some of their
derivatives. The standard numbering of the carbons on glucose is
indicated by the numbers 1 and 6. 2-KDG=2-keto-D-gluconate;
2,5-DKG=2,5-diketogluconate; 2KLG=2-keto-L-gulonate.
FIG. 10 depicts general strategy used to interrupt the gluconate
kinase gene from P. citrea.
FIG. 11 depicts the oxidative pathway for the production of
ascorbic acid. E1 stands for glucose dehydrogenase; E2 stands for
gluconic acid dehydrogenase; E3 stands for 2-keto-D-gluconic acid
dehydrogenase; and E4 stands for 2,5-diketo-D-gluconic acid
reductase.
FIG. 12 depicts the net reactions during the fermentation of host
cells capable of producing ascorbic acid intermediates.
FIG. 13 depicts carbon evolution rate (CER) and oxygen uptake rate
(OUR) of a fermentation of a wild-type organism after exposure to
glucose.
FIG. 14 depicts the CER and OUR of a fermentation with a single
delete (glucokinase).
FIG. 15 depicts the CER and OUR of a fermentation with a single
delete (gluconokinase).
FIG. 16 depicts the CER and OUR of a fermentation with a host cell
having both glucokinase and gluconokinase deleted.
FIG. 17 is a schematic illustrating the interrelationships of
various metabolic pathways (including the glycolytic pathway, TCA
cycle, and pentose pathway) and the oxidative pathways.
Glk=glucokinase; Gntk=gluconokinase; IdnO=5-keto-D-gluconate
5-reductase; IdnD=L-Idonate 5-dehydrogenase; TKT=transketolase;
TAL=transaldolase, 2KR=2-keto reductase;
2,5DKGR=2,5-diketogluconate reductase.
FIG. 18 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of ribose. The X indicate the
enzymatic steps that would be modified to effect the desired
increase in ribose yield.
FIG. 19 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of riboflavin. The X indicate the
enzymatic steps that would be modified to effect the desired
increase in ribose yield.
FIG. 20 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of nucleotides. The X indicate the
enzymatic steps that would be modified to effect the desired
increase in nucleotide yield.
FIG. 21 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of tartrate. The X indicate the
enzymatic steps that would be modified to effect the desired
increase in ribose production. IdnO=5-keto-D-gluconate 5-reductase;
IdnD=1-Idonate 5-dehydrogenase.
FIG. 22 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of gluconateribose. The X indicate
the enzymatic steps that would be modified to effect the desired
increase in gluconate production.
FIG. 23 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of erythorbic acid. The X indicate
the enzymatic steps that would be modified to effect the desired
increase in erythorbic acid production.
FIG. 24 is a schematic illustrating the interrelationships of
various central metabolic pathways and the modifications which
would increase the production of 2,5-DKG. The X indicate the
enzymatic or transport pathways that would be modified to effect
the desired increase in 2,5-diketogluconate production.
FIG. 25 is a schematic illustrating the pathway of dihydroxyacetone
phosphate (DHAP) being converted to glycerol.
FIG. 26 depicts the DNA Sequence of primers used to amplify by PCR
the 2.9 kb DNA fragment that contains the glpK gene as described in
Example 7.
FIG. 27 describes the DNA sequence of the structural gene of the
glycerol kinase from P. citrea as described in Example 7. The
sequence of the HpaI site used to interrupt the gene is
underlined.
FIG. 28 depicts the protein sequence of the glycerol kinase from P.
citrea as described in Example 7.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides methods for producing products
comprising recombinant host cells that comprise an productive
pathway and a catabolic pathway wherein said pathways are uncoupled
in the host cell during part or all of said method, that is wherein
said pathways do not compete for initial carbon source, such as
D-glucose or D-gluconic acid for example, or for cellular
components, such as co-factor and ATP during part or all of said
method. The invention encompasses methods wherein the productive
and catabolic pathways are uncoupled via modification and/or
regulation of enzymatic activities present in the productive and/or
catabolic pathways.
The invention encompasses methods wherein the productive and
catabolic pathway are coupled during part of said culturing, for
example during the early part of said culturing where it is
desirable to channel or direct host cell resources to building host
cell biomass, and uncoupled during part of said culturing, for
example, after host cell biomass has been produced or when it is
desirable to channel or direct cell resources to production of
product. The invention encompasses methods comprising culturing
recombinant host cells having a productive pathway and a catabolic
pathway that are uncoupled during all of said culturing.
The invention encompasses methods wherein the productive and
catabolic pathways are uncoupled at the stage of initial
phosphorylation of the carbon source that is used by the cell, by
modifying the genomic sequence that encodes such
phosphorylation.
The uncoupling of the productive pathway and catabolic pathway
encompasses inhibition of at least one enzymatic activity that
phosphorylates the initial carbon source and/or any carbon
metabolite in the productive and/or catabolic pathway. The
uncoupling of the productive pathway and catabolic pathway
encompasses inactivation of at least one enzymatic activity that
phosphorylates the initial carbon source and/or any carbon
metabolite in the productive and/or catabolic pathway, such as by
mutation in or deletion of part or all of the polynucleotide
encoding an enzymatic activity that phosphorylates the initial
carbon source and/or any carbon metabolite. The uncoupling of the
productive pathway and catabolic pathway encompasses regulation of
at least one enzymatic activity that phosphorylates the initial
carbon source and/or any carbon metabolite in the productive and/or
catabolic pathway.
One advantage of the invention is that in host cells comprising
uncoupled productive and catabolic pathways, the pathways are able
to function simultaneously without one pathway creating a
disadvantage for the other. In some embodiments disclosed herein, a
host cell having a deletion of glucokinase and gluconokinase is
cultured in the presence of D-glucose. The D-glucose passes through
the productive pathway without being diverted into the catabolic
pathway, thereby increasing the amount of carbon substrate
available for conversion to the desired productive pathway
generated product. Fructose, or other non-glucose carbon source,
can be fed to the host cell and is used to satisfy the host cell's
metabolic needs, freeing the D-glucose for use by the product
pathway yielding the desired product. In this embodiment, the
productive and catabolic pathways function simultaneously and
non-competitively in the host cell.
Another advantage of the invention is that in host cells comprising
uncoupled productive and catabolic pathways, either pathway can be
used to provide for the metabolic needs of the host cell, freeing
the other pathway to be used to produce products through that
particular pathway. In some embodiments disclosed herein, a host
cell having a deletion of the coupling enzymes enables the products
of the productive pathway to satisfy the metabolic needs of the
host cell, freeing the pathway usually associated with the
generation of energy through the catabolic pathway to generate
products. Thus fructose, or other non-glucose carbon source, can be
fed to the host cell and is used to produce derivatives or desired
products, while the host cell's metabolic needs are satisfied by
conversion of productive pathway products to metabolic needs in the
host cell.
In other embodiments, the ability of the host cell to use
D-glucose, or a metabolite of D-glucose, such as D-gluconate, in
the catabolic pathway, that is the ability of the host cell to
phosphorylate D-glucose or D-gluconate at their respective 6th
carbons, is regulated. Regulating the expression of the enzymatic
activity allows a process wherein D-glucose or other carbon source
is available to the catabolic pathway during the initial phase of
culturing, where it is desirable to build cell biomass, and not
available, that is not phosphorylated, at later stages of culturing
where it may be desirable to maximize ATP production for use by the
cell or where it may be desirable to feed a different carbon source
to the cell for production of desired product.
In these embodiments, a particular advantage provided by the
invention is the ability to make use of continuous fermentation
processes for the production of products.
Another advantage provided by the invention is the uncoupling of
the extracellular oxidation of a substrate from the metabolic
pathways that use those oxidation products.
Another advantage provided by the invention is the increased
efficiency in the production of products by the modified host cells
as compared to wild-type host cells as measured directly by the
increased conversion of substrate to end-product or indirectly as
measured by O2 consumption or CO.sub.2 production.
A further advantage provided by the invention is the ability of the
host cell to utilize two different carbon sources simultaneously
for the production of products.
General Techniques
The practice of the present invention will employ, unless otherwise
indicated, conventional techniques of molecular biology (including
recombinant techniques), microbiology, cell biology, and
biochemistry, which are within the skill of the art. Such
techniques are explained fully in the literature, such as,
Molecular Cloning: A Laboratory Manual, second edition (Sambrook et
al., 1989); Current Protocols in Molecular Biology (F. M. Ausubel
et al., eds., 1987 and annual updates); Oligonucleotide Synthesis
(M. J. Gait, ed., 1984); and PCR: The Polymerase Chain Reaction,
(Mullis et al., eds., 1994). Manual of Industrial Microbiology and
Biotechnology, Second Edition (A. L. Demain, et al., eds. 1999)
Definitions
As used herein, the term "uncoupled" when referring to productive
and catabolic pathways of a host cell means that the productive
pathway of the host cell, including the substrates and products
produced therein, have a reduced diversion of substrates to the
catabolic pathway of the host cell. By reduced diversion, is meant
that the yield of the wild type is less than the yield of the
modified host cell.
As used herein, "productive pathway of a host cell" means that the
host cell comprises at least one enzyme that coverts a carbon
source, such as, D-glucose and/or its metabolites to a desired
product or intermediate. The productive pathway of the host cell
includes but is not limited to the oxidative pathway of the host
cell.
As used herein, "oxidative pathway of a host cell" means that the
host cell comprises at least one-oxidative enzyme that oxidizes a
carbon source, such as, D-glucose and/or its metabolites. A
"membrane" or "membrane bound" glucose productive pathway in a host
cells refers to a host cell that oxidizes a carbon source such as,
D-glucose and/or its metabolites, via at least one membrane bound
productive enzymatic activity. In some embodiments, an oxidative
pathway in a host cell comprises one enzymatic activity. In other
embodiments, an oxidative pathway in a host cell comprises two or
more enzymatic activities.
As used herein, "catabolic pathway of a host cell" means that the
host cell comprises at least one enzymatic activity that generates
ATP or NADPH, for example, by phosphorylating a carbon source, such
as D-glucose and/or its metabolites. An "intracellular" catabolic
pathway in a host cell means that the host cell comprises at least
one such enzymatic activity in the host cell cytosol. In some
embodiments, a catabolic pathway in a host cell comprises one
enzymatic activity. In other embodiments, a catabolic pathway in a
host cell comprises two or more enzymatic activities.
As used herein, the phrase "enzymatic activity which phosphorylates
D-glucose at its 6th carbon" refers to an enzymatic activity that
adds a phosphate to the 6th carbon of D-glucose and includes the
enzymatic activities glucokinase (EC-2.7.1.2); and
phosphotransferase system (PTS) (E.C.-2.7.1.69). As used herein,
the phrase "enzymatic activity which phosphorylates D-gluconate at
its 6th carbon" refers to an enzymatic activity that phosphorylates
D-gluconate at its 6th carbon and includes the enzymatic activity
gluconokinase (E.C.-2.7.1.12).
As used herein, "modifying" the levels of an enzymatic activity
produced by a host cell or "modified levels" of an enzymatic
activity of a host cell refers to controlling the levels of
enzymatic activity produced during culturing, such that the levels
are increased or decreased as desired. The desired change in the
levels of enzymatic activity may be genetically engineered to take
place in one or both enzymatic activities either simultaneously or
sequentially, in any order. In order to control the levels of
enzymatic activity, the host cell is genetically engineering such
that nucleic acid encoding the enzymatic activity is
transcriptionally or translationally controlled.
As used herein, the term "modified" when referring to nucleic acid
or polynucleotide means that the nucleic acid has been altered in
some way as compared to wild type nucleic acid, such as by mutation
in; deletion of part or all of the nucleic acid; or by being
operably linked to a transcriptional control region. As used herein
the term "mutation" when referring to a nucleic acid refers to any
alteration in a nucleic acid such that the product of that nucleic
acid is partially or totally inactivated or eliminated. Examples of
mutations include but are not limited to point mutations, frame
shift mutations and deletions of part or all of a gene encoding an
enzymatic activity, such as an enzymatic activity that transports
the substrate across the cell membrane, e.g., phosphorylates
D-glucose at its 6th carbon or an enzymatic activity that
phosphorylates D-gluconate at its 6th carbon.
An "altered bacterial strain" according to the invention is a
genetically engineered bacterial microorganism having an enhanced
level of production over the level of production of the same
end-product in a corresponding unaltered bacterial host strain
grown under essentially the same growth conditions. An "unaltered
bacterial strain" or host is a bacterial microorganism wherein the
coding sequence of the diverting enzymatic pathway is not
inactivated and remains enzymatically active. The enhanced level of
production results from the inactivation of one or more chromosomal
genes. In a first embodiment the enhanced level of expression
results from the deletion of one or more chromosomal genes. In a
second embodiment the enhanced level of expression results from the
insertional inactivation of one or more chromosomal genes.
Preferably the inactivated genes are selected from those encoding
the enzymes whose inactivity is desired as described elsewhere in
this application. For example, in one embodiment one or more
chromosomal genes is selected from the group consisting of glk, and
gntk.
In certain embodiments, the altered bacterial strain may embody two
inactivated genes, three inactivated genes, four inactivated genes,
five inactivated genes, six inactivated genes or more. The
inactivated genes may be contiguous to one another or may be
located in separate regions of the chromosome. An inactivated
chromosomal gene may have a necessary function under certain
conditions, but the gene is not necessary for microorganism strain
viability under laboratory conditions. Preferred laboratory
conditions include but are not limited to conditions such as growth
in a fermentator, in a shake plate, in plate media or the like.
As used herein, the term "inactivation" or "inactivating" when
referring to an enzymatic activity means that the activity has been
eliminated by any means including a mutation in or deletion of part
or all of the nucleic acid encoding the enzymatic activity. The
term "inactivation" or "inactivating" includes any method that
prevents the functional expression of one or more of the desired
chromosomal genes, wherein the gene or gene product is unable to
exert its known function. The desired chromosomal genes will depend
upon the enzymatic activity that is intended to be inactivated. For
example the inactivation of glucokinase and/or gluconokinase
activity can be effected by inactivating the glk and/or gntk
chromosomal genes coding regions. Inactivation may include such
methods as deletions, mutations, interruptions or insertions in the
nucleic acid gene sequence. In one embodiment, the expression
product of an inactivated gene may be a truncated protein as long
as the truncated protein does not show the biological activity of
the unaltered coding region. In an altered bacterial strain
according to the invention, the inactivation of the one or more
genes will preferably be a stable and non-reverting
inactivation.
In a preferred embodiment, preferably a gene is deleted by
homologous recombination. For example, as shown in FIG. 9, when glk
is the gene to be deleted, a chloramphenicol resistance gene is
cloned into a unique restriction site found in the glucokinase
gene. The Cm.sup.R gene is inserted into the structural coding
region of the gene at the Pst I site. Modification is then
transferred to the chromosome of a P. citrea glkA- by homologous
recombination using a non-repliation R6K vector. The Cm.sup.R gene
is subsequently removed from the glk coding region leaving an
interrupting spacer in the coding region, inactivating the coding
region. In another embodiment, the Cm.sup.R gene is inserted into
the coding region in exchange for portions of the coding region.
Subsequent removal of the Cm.sup.R gene without concomitant return
of the exchanged out portion of the coding region results in an
effective deletion of a portion of the coding region, inactivating
such region.
A deletion of a gene as used herein may include deletion of the
entire coding sequence, deletion of part of the coding sequence, or
deletion of the coding sequence including flanking regions. The
deletion may be partial as long as the sequences left in the
chromosome are too short for biological activity of the gene. The
flanking regions of the coding sequence may include from about 1 bp
to about 500 bp at the 5' and 3' ends. The flanking region may be
larger than 500 bp but will preferably not include other genes in
the region which may be inactivated or deleted according to the
invention. The end result is that the deleted gene is effectively
non-functional.
In another preferred embodiment, inactivation is by insertion. For
example when glk is the gene to be inactivated, a DNA construct
will comprise an incoming sequence having the glk gene interrupted
by a selective marker. The selective marker will be flanked on each
side by sections of the glk coding sequence. The DNA construct
aligns with essentially identical sequences of the glk gene in the
host chromosome and in a double crossover event the glk gene is
inactivated by the insertion of the selective marker.
In another embodiment, inactivation is by insertion in a single
crossover event with a plasmid as the vector. For example, a glk
chromosomal gene is aligned with a plasmid comprising the gene or
part of the gene coding sequence and a selective marker. The
selective marker may be located within the gene coding sequence or
on a part of the plasmid separate from the gene. The vector is
integrated into the Bacillus chromosome, and the gene is
inactivated by the insertion of the vector in the coding
sequence.
Inactivation may also occur by a mutation of the gene. Methods of
mutating genes are well known in the art and include but are not
limited to chemical mutagenesis, site-directed mutation, generation
of random mutations, and gapped-duplex approaches. (U.S. Pat. No.
4,760,025; Moring et al., Biotech. 2:646 (1984); and Kramer et al.,
Nucleic Acids Res. 12:9441 (1984)).
Inactivation may also occur by applying the above described
inactivation methods to the respective promoter regions of the
desired genomic region.
"Under transcriptional control" or "transcriptionally controlled"
are terms well understood in the art that indicate that
transcription of a polynucleotide sequence, usually a DNA sequence,
depends on its being operably (operatively) linked to an element
which contributes to the initiation of, or promotes, transcription.
"Operably linked" refers to a juxtaposition wherein the elements
are in an arrangement allowing them to function.
As used herein, the term "regulatable promoter" refers o a promoter
element which activity or function can be modulated. This
modulation can be accomplished in many different ways, most
commonly by the interaction of protein(s) that interfere or
increase the ability of the RNA polymerase enzyme to initiate
transcription.
"Under translational control" well understood in the art that
indicates a regulatory process that occurs after the messenger RNA
has been formed.
As used herein, the term "batch" describes a batch cell culture to
which substrate, in either solid or concentrated liquid form, is
added initially at the start of the run. A batch culture is
initiated by inoculating cells to the medium, but, in contrast to a
fed-batch culture, there is no subsequent inflow of nutrients, such
as by way of a concentrated nutrient feed. In contrast to a
continuous culture, in a batch cell culture, there is no systematic
addition or systematic removal of culture fluid or cells from a
culture. There is no ability to subsequently add various analytes
to the culture medium, since the concentrations of nutrients and
metabolites in culture medium are dependent upon the initial
concentrations within the batch and the subsequent alteration of
the composition of the nutrient feed due to the act of
fermentation.
As used herein, the term "fed-batch" describes a batch cell culture
to which substrate, in either solid or concentrated liquid form, is
added either periodically or continuously during the run. Just as
in a batch culture, a fed-batch culture is initiated by inoculating
cells to the medium, but, in contrast to a batch culture, there is
a subsequent inflow of nutrients, such as by way of a concentrated
nutrient feed. In contrast to a continuous culture there is no
systematic removal of culture fluid or cells from a fed-batch
culture is advantageous in applications that involve monitoring and
manipulating the levels of various analytes in the culture medium,
since the concentrations of nutrients and metabolites in culture
medium can be readily controlled or affected by altering the
composition of the nutrient feed. The nutrient feed delivered to a
fed-batch culture is typically a concentrated nutrient solution
containing an energy source, e.g., carbohydrates; optionally, the
concentrated nutrient solution delivered to a fed-batch culture can
contain amino acids, lipid precursors and/or salts. In a fed-batch
culture, this nutrient feed is typically rather concentrated to
minimize the increase in culture volume while supplying sufficient
nutrients for continued cell growth.
The term "continuous cell culture" or, simply, "continuous culture"
is used herein to describe a culture characterized by both a
continuous inflow of a liquid nutrient feed and a continuous liquid
outflow. The nutrient feed may, but need not, be a concentrated
nutrient feed. Continuously supplying a nutrient solution at about
the same rate that cells are washed out of the reactor by spent
medium allows maintenance of a culture in a condition of stable
multiplication and growth. In a type of bioreactor known as a
chemostat, the cell culture is continuously fed fresh nutrient
medium, and spent medium, cells and excreted cell product are
continuously drawn off. Alternatively, a continuous culture may
constitute a "perfusion culture," in which case the liquid outflow
contains culture medium that is substantially free of cells, or
substantially lower cell concentration than that in the bioreactor.
In a perfusion culture, cells can be retained by, for example,
filtration, centrifugation, or sedimentation.
"Culturing" as used herein refers to fermentive bioconversion of a
carbon substrate to the desired end-product within a reactor
vessel. Bioconversion as used herein refers to the use of
contacting a microorganism with the carbon substrate to convert the
carbon substrate to the desired end-product.
As used herein, "Oxygen Uptake Rate or "OUR" refers to the
determination of the specific consumption of oxygen within the
reactor vessel. Oxygen consumption can be determined using various
on-line measurements. In one example, the OUR (mmol/(liter*hour))
is determined by the following formula: ((Airflow (standing liters
per minute)/Fermentation weight (weight of the fermentation broth
in kilograms)).times.supply O.sub.2.times.broth density.times.(a
constant to correct for airflow calibration at 21.1 C instead of
standard 20.0 C)) minus ([airflow/fementation weight].times.[offgas
O.sub.2/offgas N.sub.2].times.supply N.sub.2.times.broth
density.times.constant).
As used herein, "carbon evolution rate or "CER" refers to the
determination of how much CO.sub.2 is produced within the reactor
vessel during fermentation. Usually, since no CO.sub.2 is initially
or subsequently provided to the reaction vessel, any CO.sub.2 is
assumed to be produced by the fermentation process occurring within
the reaction vessel. "Off-gas CO.sub.2" refers to the amount of
CO.sub.2 measured within the reactor vessel, usually by mass
spectroscopic methods known in the art.
As used herein, "yield" refers to the amount of product divided by
the amount of substrate. The yield can be expressed as a weight %
(product gm/substrate gm) or as moles of product/moles of
substrate. For example, the amount of the substrate, e.g., glucose
can be determined by the feed rate and the concentration of the
added glucose. The amount of products present can be determined by
various spectrophotometric or analytic methodologies. One such
methodology is high performance liquid chromatography (HPLC). An
increased yield refers to an increased yield as compared to the
yield of a conversion using the wild-type organism, for example an
increase of 10%, 20%, or 30% over the yield of the wild-type.
The phrase "oxidative enzyme" as used herein refers to an enzyme or
enzyme system which can catalyze conversion of a substrate of a
given oxidation state to a product of a higher oxidation state than
substrate. The phrase "reducing enzyme" refers to an enzyme or
enzyme system which can catalyze conversion of a substrate of a
given oxidation state to a product of a lower oxidation state than
substrate. In one illustrative example disclosed herein, productive
enzymes-associated with the biocatalysis of D-glucose or its
metabolites in a Pantoea cell which has been engineered to produce
ASA intermediates, include among others D-glucose dehydrogenase,
D-gluconate dehydrogenase and 2-keto-D-gluconate dehydrogenase. In
another illustrative embodiment disclosed herein, reducing enzymes
associated with the biocatalysis of D-glucose or its metabolites in
a Pantoea cell which has been engineered to produce ASA
intermediates, as described herein, include among others
2,5-diketo-D-gluconate reductase, 2-keto reductase and 5-keto
reductase. Such enzymes include those produced naturally by the
host strain or those introduced via recombinant means.
As used herein, the term carbon source encompasses suitable carbon
sources ordinarily used by microorganisms, such as 6 carbon sugars,
including but not limited to glucose, gulose, sorbose, fructose,
idose, galactose and mannose all in either D or L form, or a
combination of 6 carbon sugars, such as glucose and fructose,
and/or 6 carbon sugar acids including but not limited to
2-keto-L-gulonic acid, idonic acid, gluconic acid,
6-phosphogluconate, 2-keto-D-gluconic acid, 5-keto-D-gluconic acid,
2-ketogluconatephosphate, 2,5-diketo-L-gulonic acid,
2,3-L-diketogulonic acid, dehydroascorbic acid, erythroascorbic
acid, erythorbic acid and D-mannonic acid or the enzymatic
derivatives of such.
The following definitions apply as used herein to D-glucose or
glucose (G); D-gluconate or gluconate (GA); 2-keto-D-gluconate
(2KDG); 2,5-diketo-D-gluconate (2,5DKG or DKG); 2-keto-L-gulonic
acid (2KLG, or KLG); L-idonic acid (IA); erythorbic acid (EA);
ascorbic acid (ASA); glucose dehydrogenase (GDH); gluconic acid
dehydrogenase (GADH); 2,5-diketo-D-gluconate reductase (DKGR);
2-keto-D-gluconate reductase (KDGDH); D-ribose (R); 2-keto
reductase (2KR or KR); and 5-keto reductase (5KR or KR).
"Carbon metabolite" as used herein refers to a compound that is
utilized in the catabolic pathway to generate ATP, NADPH and/or is
phosphorylated for transport into the cell.
"Allowing the production of an ascorbic acid intermediate from the
carbon source, wherein the production of said ascorbic acid
intermediate is enhanced compared to the production of the ascorbic
acid intermediate in the unaltered bacterial host strain" means
contacting the substrate, e.g. carbon source, with the altered
bacterial strain to produce the desired end-product. The inventors
discovered that by altering certain enzymatic activities by
inactivating genomic expression, the microorganism demonstrated
enhanced end-product production.
"Desired end-product" as used herein refers to the desired compound
to which the carbon substrate is bioconverted into. The desired
end-product may be the actual compound sought or an intermediate
along another pathway. Exemplary desired end-products are listed on
the right side of FIG. 3.
As used herein, the term "bacteria" refers to any group of
microscopic organisms that are prokaryotic, i.e., that lack a
membrane-bound nucleus and organelles. All bacteria are surrounded
by a lipid membrane that regulates the flow of materials in and out
of the cell. A rigid cell wall completely surrounds the bacterium
and lies outside the membrane. There are many different types of
bacteria, some of which include, and are not limited to those
strains within the families of Enterobacteriaceae, Bacillus,
Streptomyces, Pseudomonas, and Erwinia.
As used herein, the family "Enterobacteriaceae" refers to bacterial
strains having the general characteristics of being Gram negative
and being facultatively anaerobic. For the production of ASA
intermediates, preferred Enterobacteriaceae strains are those that
are able to produce 2,5-diketo-D-gluconic acid from D-glucose or
carbon sources which can be converted to D-glucose by the strain.
Included in the family of Enterobacteriaceae which are able to
produce 2,5-diketo-D-gluconic acid from D-glucose solutions are the
genus Erwinia, Enterobacter, Gluconobacter and Pantoea, for
example. Intermediates in the microbial pathway from carbon source
to ASA, include but are not limited to GA, KDG, DKG, DKG, KLG and
IA. In the present invention, a preferred Enterobacteriaceae
fermentation strain for the production of ASA intermediates is a
Pantoea species and in particular, Pantoea citrea.
As used herein the family "Bacillus" refers to rod-shaped bacterial
strains having the general characteristics of being gram positive,
capable of producing spores under certain environmental conditions.
Other Enterobacteriaceae strains that produce ASA intermediates
include, but are not limited to, E. coli and Gluconobacter.
As used herein, the term "recombinant" refers to a host cell that
has a modification of its genome, eg as by the additional of
nucleic acid not naturally occurring in the organism or by a
modification of nucleic acid naturally occurring in the host cell
and includes host cells having additional copies of endogenous
nucleic acid introduced via recombinant means. The term
"heterologous" as used herein refers to nucleic acid or amino acid
sequences not naturally occurring in the host cell. As used herein,
the term "endogenous" refers to a nucleic acid naturally occurring
in the host.
The terms "isolated" or "purified" as used herein refer to an
enzyme, or nucleic acid or protein or peptide or co-factor that is
removed from at least one component with which it is naturally
associated. In the present invention, an isolated nucleic acid can
include a vector comprising the nucleic acid.
It is well understood in the art that the acidic derivatives of
saccharides, may exist in a variety of ionization states depending
upon their surrounding media, if in solution, or out of solution
from which they are prepared if in solid form. The use of a term,
such as, for example, idonic acid, to designate such molecules is
intended to include all ionization states of the organic molecule
referred to. Thus, for example, "idonic acid", its cyclized form
"idonolactone", and "idonate" refer to the same organic moiety, and
are not intended to specify particular ionization states or
chemical forms.
As used herein, the term "vector" refers to a polynucleotide
construct designed for transduction/transfection of one or more
cell types including for example, "cloning vectors" which are
designed for isolation, propagation and replication of inserted
nucleotides or "expression vectors" which are designed for
expression of a nucleotide sequence in a host cell, such as a
Pantoea citrea or E. coli host cell.
The terms "polynucleotide" and "nucleic acid", used interchangeably
herein, refer to a polymeric form of nucleotides of any length,
either ribonucleotides or deoxyribonucleotides. These terms include
a single-, double- or triple-stranded DNA, genomic DNA, cDNA, RNA,
DNA-RNA hybrid, or a polymer comprising purine and pyrimidine
bases, or other natural, chemically, biochemically modified,
non-natural or derivatized nucleotide bases. The backbone of the
polynucleotide can comprise sugars and phosphate groups (as may
typically be found in RNA or DNA), or modified or substituted sugar
or phosphate groups. Alternatively, the backbone of the
polynucleotide can comprise a polymer of synthetic subunits such as
phosphoramidates and thus can be a oligodeoxynucleoside
phosphoramidate (P--NH2) or a mixed phosphoramidate-phosphodiester
oligomer. Peyrottes et al. (1996) Nucleic Acids Res. 24: 1841-8;
Chaturvedi et al. (1996) Nucleic Acids Res. 24: 2318-23; Schultz et
al. (1996) Nucleic Acids Res. 24: 2966-73. A phosphorothioate
linkage can be used in place of a phosphodiester linkage. Braun et
al. (1988) J. Immunol. 141: 2084-9; Latimer et al. (1995) Molec.
Immunol. 32: 1057-1064. In addition, a double-stranded
polynucleotide can be obtained from the single stranded
polynucleotide product of chemical synthesis either by synthesizing
the complementary strand and annealing the strands under
appropriate conditions, or by synthesizing the complementary strand
de novo using a DNA polymerase with an appropriate primer.
Reference to a polynucleotide sequence (such as referring to a SEQ
ID NO) also includes the complement sequence.
The following are non-limiting examples of polynucleotides: a gene
or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes,
cDNA, recombinant polynucleotides, branched polynucleotides,
plasmids, vectors, isolated DNA of any sequence, isolated RNA of
any sequence, nucleic acid probes, and primers. A polynucleotide
may comprise modified nucleotides, such as methylated nucleotides
and nucleotide analogs, uracyl, other sugars and linking groups
such as fluororibose and thioate, and nucleotide branches. The
sequence of nucleotides may be interrupted by non-nucleotide
components. A polynucleotide may be further modified after
polymerization, such as by conjugation with a labeling component.
Other types of modifications included in this definition are caps,
substitution of one or more of the naturally occurring nucleotides
with an analog, and introduction of means for attaching the
polynucleotide to proteins, metal ions, labeling components, other
polynucleotides, or a solid support. Preferably, the polynucleotide
is DNA. As used herein, "DNA" includes not only bases A, T, C, and
G, but also includes any of their analogs or modified forms of
these bases, such as methylated nucleotides, internucleotide
modifications such as uncharged linkages and thioates, use of sugar
analogs, and modified and/or alternative backbone structures, such
as polyamides.
A polynucleotide or polynucleotide region has a certain percentage
(for example, 80%, 85%, 90%, 95%, 97% or 99%) of "sequence
identity" to another sequence means that, when aligned, that
percentage of bases are the same in comparing the two sequences.
This alignment and the percent homology or sequence identity can be
determined using software programs known in the art, for example
those described in Current Protocols in Molecular Biology (F. M.
Ausubel et al., eds., 1987) Supplement 30, section 7.7.18. A
preferred alignment program is ALIGN Plus (Scientific and
Educational Software, Pennsylvania), preferably using default
parameters, which are as follows: mismatch=2; open gap=0; extend
gap=2.
A polynucleotide sequence that is "depicted in" a SEQ ID NO means
that the sequence is present as an identical contiguous sequence in
the SEQ ID NO. The term encompasses portions, or regions of the SEQ
ID NO as well as the entire sequence contained within the SEQ ID
NO.
"Expression" includes transcription and/or translation.
As used herein, the term "comprising" and its cognates are used in
their inclusive sense; that is, equivalent to the term "including"
and its corresponding cognates.
"A," "an" and "the" include plural references unless the context
clearly dictates otherwise.
Productive and Catabolic Pathways of Host Cells
FIGS. 2 and 3 describe some of the products of metabolism that can
be obtained from some of the metabolic routes. The majority of the
products on the left side of FIG. 3 (Glucose-6-phosphate,
glucose-1-phosphate; fructose-6-phosphate, mannose-6-phosphate,
dihydroacetone-phosphate; dihydroacetone; glycerol;
1,2-propanediol; 1,3 propanediol; lactic acid; succinic acid;
oxalic acid; citric acid; fumaric acid; malic acid; amino acids;
glycogen; trehalose; and UDP-glucose) can be obtained from the
catabolic or TCA cycle. On the contrary, the compounds on the
right, desired end-products for the purposes of this invention
(gluconic acid, 2-keto-D-gluconic acid, 2,5-di-keto-gluconate;
erythorbic acid; 5-keto-D-gluconate; tartaric acid; D-ribose;
riboflavin; deoxyribonucleotides; aromatic amino acids, aromatic
compounds [e.g. P-hydroxybenzoic acid; quinines; catechols;
indoles; indigo; gallic acid; pyrogallol; melanin, adipic acid,
p-aminobenzoic acid]; pyridoxine and aspartame) derive most of its
carbon from the pentose pathway and/or from the oxidation of
glucose into keto acid. In many cases, these products are not
natural products of the metabolism of a particular cell, but they
can be produced by adding or removing certain enzymatic
functions.
Generally, those products on the left side of FIG. 3 are used to
maintain the catabolic needs of the host cell. By uncoupling the
interaction between those compounds on the left with those on the
right, the metabolic requirements of the host cell are satisfied by
the products generated on one side, enabling more carbon substrate
to be converted into the desired productive product. In one
embodiment, the uncoupling of the productive pathways from the
catabolic pathways increase the yield of compounds produced on the
right side. In another embodiment, it is contemplated using the
products generated by the productive pathways to maintain the
metabolic requirements of the host cell-would enable those
reactions in the catabolic pathways to be utilized to increase the
yield of products derived from those products within the catabolic
pathway, e.g. 1,3-propanediol, DHAP, lactic acid.
The invention also includes functionally-preserved variants of the
modified nucleic acid sequences disclosed herein, which include
nucleic acid substitutions, additions, and/or deletions. In one
embodiment, the variants include modified sequences encoding
glucokinase and gluconokinase, which inactivates the enzymatic
pathway converting glucose to glucose-6-phosphate and gluconate to
gluconate-6-phosphate, uncoupling the productive pathways from the
catabolic pathways, reducing the amount of carbon substrate
diverted to the catabolic pathway and increasing the amount of
carbon substrate available for conversion into the desired product,
e.g. 2-KLG. Genetic modifications are used to eliminate the
communication between the catabolic functions and the enzymatic
reactions that are required to synthesize a desired product. While
various modifications are described in this application (see FIGS.
17-24), the inventors contemplate that other enzymatic steps could
be modified to achieve the same uncoupling oxidative, catabolic
pathway uncoupling.
Esters of phosphoric acid are encountered with trioses, tetroses,
pentoses, hexoses and heptoses. The phosphorylation of all sugars
is the initial step in their metabolism. Thus glucose can be
phosphorylated to glucose 6-phospahte. All cells that can
metabolize glucose contain some form of a hexokinase which catalyze
the reaction
##STR00001## FIG. 9 depicts D-glucose and illustrates the "6th
carbon". Exemplary hexokinases include hexokinase (Frohlich, et
al., 1985, Gene 36:105-111) and glucokinase (Fukuda, et al., 1983,
J. Bacteriol. 156:922-925). The DNA sequence of the glucokinase
structural gene from P. citrea is shown in FIG. 4. The recogition
site for the restriction enzymes NcoI (CCATGG) and SnaBI (TACGTA)
are highlighted. FIG. 5 depicts the protein sequence of the
glucokinase gene from P. citrea. Most hexokinases are somewhat
nonspecific, showing some ability to catalyze formation of
6-phosphate esters of mannose, fructose, and galactose. In
addition, other hexose derivatives may also be phosphorylated by a
hexokinase. Gluconate (FIG. 3), for example, may also be
phosphorylated by a kinase, specifically gluconokinase (citation).
The sequence for the gluconokinase structural gene from P. citrea
is depicted in FIG. 6. The recognition site for the restriction
enzyme PstI (CTGCAG) is highlighted. The protein sequence for the
gluconokinase gene from P. citrea is depicted in FIG. 7 (SEQ ID NO
4). The some of the genes for glucokinase And gluconokinase (glk,
gntk, etc.) are shown in FIG. 8.
FIG. 17 shows the interrelationships between the catabolic pathways
and the productive (oxidative) pathway. Glucose can enter the
catabolic pathways through the glycolytic pathway by the
phosphorylation of glucose to glucose-6-phosphate by glucokinase
(Glk); and through the pentose pathway by the phosphorylation of
gluconate to gluconate-6-phosphate by glucono kinase (Gntk).
Inactivation or modifying the levels of glucokinase and
gluconokinase by modifying the nucleic acid or polypeptide encoding
the same (glk or gntk), results in the increased yield of the
desired product, e.g. an ascorbic acid intermediate. As used
herein, ascorbic acid intermediate includes those sugar acids
produced within the oxidative pathway from glucose to 2KLG,
including, but not limited to gluconate, 2-KGD, 2,5-DKG, 2-KLG, and
5-DKG.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of ribose. As shown
in FIG. 18, glucose can enter the catabolic pathway through the
glycolytic pathway, for example through glucose-6-phosphate,
fructose-6-phosphate, and/or glyceraldehydes-3-phosphate.
Inactivation or modifying the levels of glucokinase, gluconokinase,
ribulose-5-phosphate epimerase, transketolase and transaldolase, by
modifying the nucleic acid or polypeptide encoding the same,
results in the increased yield of the desired product, e.g.
ribose.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of riboflavin. As
shown in FIG. 19, glucose can enter the catabolic pathway through
the glycolytic pathway, for example through glucose-6-phosphate,
and the pentose pathway. Inactivation or modifying the levels of
glucokinase, ribulose-5-phosphate epimerase and ribose-5-phosphate
isomerase, by modifying the nucleic acid or polypeptide encoding
the same, results in the increased yield of the desired product,
e.g. riboflavin.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of nucleotides. As
shown in FIG. 20, glucose can enter the catabolic pathway through
the glycolytic pathway, for example through glucose-6-phosphate,
fructose-6-phosphate, and/or glyceraldehydes-3-phosphate.
Inactivation or modifying the levels of glucokinase,
ribulose-5-phosphate epimerase, transaldolase and transketolase, by
modifying the nucleic acid or polypeptide encoding the same,
results in the increased yield of the desired product, e.g.
nucleotides.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of 5-KDG and/or
tartrate. As shown in FIG. 21, glucose can enter the catabolic
pathway through the glycolytic pathway, for example through
glucose-6-phosphate, the pentose pathway through
gluconate-6-phosphate, and other ascorbic acid by-products, such as
idonate and 2-KLG. Inactivation or modifying the levels of
glucokinase, gluconokinase, 2,5-DKG reductase, and Idonate
dehydrogenase, by modifying the nucleic acid or polypeptide
encoding the same, results in the increased yield of the desired
product, e.g. 5-DKG and/or tartrate.
In another embodiment, the catabolic pathway is Uncoupled from the
productive pathway to increase the production of gluconate. As
shown in FIG. 22, glucose can enter the catabolic pathway through
the glycolytic pathway, for example through glucose-6-phosphate and
the pentose pathway, through gluconate-6-phosphate. Inactivation or
modifying the levels of glucokinase, gluconokinase, and
glyceraldhehyde hydrogenase, by modifying the nucleic acid or
polypeptide encoding the same, results in the increased yield of
the desired product, e.g. gluconate.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of erythorbic acid.
As shown in FIG. 23, glucose can enter the catabolic pathway
through the glycolytic pathway, for example through
glucose-6-phosphate; the pentose pathway, through
gluconate-6-phosphate; and by an enzymatic transport system
transporting 2-KDG and 2,5-KDG into the cytoplasm. Inactivation or
modifying the levels of glucokinase, gluconokinase, glyceraldhehyde
hydrogenase and the transport system of 2-KDG into the cytoplasm,
by modifying the nucleic acid or polypeptide encoding the same,
results in the increased yield of the desired product, e.g.
erythoric acid.
In another embodiment, the catabolic pathway is uncoupled from the
productive pathway to increase the production of 2,5-DKG. As shown
in FIG. 24, glucose can enter the catabolic pathway through the
glycolytic pathway, for example through glucose-6-phosphate; the
pentose pathway, through gluconate-6-phosphate; and by an enzymatic
transport system transporting 2-KDG and 2,5-KDG into the cytoplasm.
Inactivation or modifying the levels of glucokinase, gluconokinase,
and 2-KDG hydrogenase; and the enzymatic transport system for
2-KDG, by modifying the nucleic acid or polypeptide encoding the
same, results in the increased yield of the desired product, e.g.
2,5-DKG. Wherein the inventors have provided in some instances
amino acid sequences and nucleotide sequences for genomic coding
regions and/or protein (enzymes) in question, those skilled in the
art will recognize that the genomic loci not specifically provided
herein are readily ascertainable by construction of probes or
hybridizing sequences incorporating already known sequences and a
homology alignment of (for example BLAST), in one embodiment, at
least 30% or at least 50%, another embodiment, of the known coding
region sequence. In another embodiment, a homology alignment of at
least 60%, 70&, 75%, 80%, 90%, 95%, 97% or even 98% of the
known sequence will identify the coding region to which the
inactivation techniques described elsewhere are applied to effect
the desired. Another methodology to determine the coding regions
for the particular enzyme known to those of skill in the art is to
obtain several known sequences, align the sequences to determine
the conserved region, then design degenerate oligoprimers followed
by PCR amplification of the connecting regions between the framing
residues to ascertain the desired genomic region.
The availability of recombinant techniques to effect expression of
enzymes in foreign hosts permits the achievement of the aspect of
the invention which envisions production of a desired end-product,
e.g., riboflavin, tartrate, 5-KDG, ribose, nucleotides, gluconate,
erythorbic acid, 2,5-DKG, other ascorbic acid intermediates or
other desired products with a reduced amount of carbon substrate
diverted to catabolic pathways from a readily available carbon
substrate. This method has considerable advantage over presently
used methods in characterized by a reduction in the amount of
substrate converted to the catabolic pathway and thus unavailable
for conversion to the desired oxidative end-product, e.g., an
ascorbic acid intermediate. This results in increased fermentative
efficiency and increased yield over fermentations with wild type
organisms. Certain wild type organisms may produce ascorbic acid
intermediates, e.g., 2-KLG, however the level produced may not be
sufficient to be economically practical. It has been observed that
wild type Pantoea citrea has its own cytoplasmic glucokinase and
gluconokinase enabling the organism to convert glucose to
phosphorylated derivatives for use in its central metabolic
pathways and the production of which, necessarily consume energy,
ATP and causes that more carbon goes to non-2-KLG producing
pathways. Under the same controlled conditions and using the method
of this invention, described below, in two interruption plasmid
described elsewhere in this application the glucokinase and
gluconokinase genes can be deleted from the P. citrea genome,
enabling the modified P. citrea to produce increased DKG from
glucose at a a level increased over the wild-type, e.g. level of
63% yield to about 97-98% yield. [see Example 6].
Other variants include, but are not limited to, inactivations of
the gap gene to increase production of dihydroacetone-phosphate,
DHAP; erythorbic acid; and tartic acid.
The variants of the sequences disclosed herein may be 80%, 85%,
90%, 95%, 98%, 99% or more identical, as measured by, for example,
ALIGN Plus (Scientific and Educational Software, Pennsylvania),
preferably using default parameters, which are as follows:
mismatch=2; open gap=0; extend gap=2 to any of the enzymatic
sequences disclosed herein. Variants of glucokinase and
gluconokinase sequences may also hybridize at high stringency, that
is at 68.degree. C. and 0.1.times.SSC, to the glucokinase and
gluconokinase sequences disclosed herein.
In terms of hybridization conditions, the higher the sequence
identity required, the more stringent are the hybridization
conditions if such sequences are determined by their ability to
hybridize to a sequence of SEQ ID NO:1 or SEQ ID NO:3. Accordingly,
the invention also includes polynucleotides that are able to
hybridize to a sequence comprising at least about 15 contiguous
nucleotides (or more, such as about 25, 35, 50, 75 or 100
contiguous nucleotides) of SEQ ID NO:1 or SEQ ID NO:3. The
hybridization conditions would be stringent, i.e., 80.degree. C.
(or higher temperature) and 6M SSC (or less concentrated SSC).
Another set of stringent hybridization conditions is 68.degree. C.
and 0.1.times.SSC. For discussion regarding hybridization
reactions, see below.
Hybridization reactions can be performed under conditions of
different "stringency". Conditions that increase stringency of a
hybridization reaction of widely known and published in the art.
See, for example, Sambrook et al. (1989) at page 7.52. Examples of
relevant conditions include (in order of increasing stringency):
incubation temperatures of 25.degree. C., 37.degree. C., 50.degree.
C. and 68.degree. C.; buffer concentrations of 10.times.SSC,
6.times.SSC, 1.times.SSC, 0.1.times.SSC (where SSC is 0.15 M NaCl
and 15 mM citrate buffer) and their equivalents using other buffer
systems; formamide concentrations of 0%, 25%, 50%, and 75%;
incubation times from 5 minutes to 24 hours; 1, 2, or more washing
steps; wash incubation times of 1, 2, or 15 minutes; and wash
solutions of 6.times.SSC, 1.times.SSC, 0.1.times.SSC, or deionized
water. An exemplary set of stringent hybridization conditions is
68.degree. C. and 0.1.times.SSC.
"T.sub.m" is the temperature in degrees Celcius at which 50% of a
polynucleotide duplex made of complementary strands hydrogen bonded
in anti-parallel direction by Watson-Crick base pairing dissociates
into single strands under conditions of the experiment. T.sub.m may
be predicted according to a standard formula, such as:
T.sub.m=81.5+16.6 log[X.sup.+]+0.41(% G/C)-0.61 (% F)-600/L where
[X.sup.+] is the cation concentration (usually sodium ion,
Na.sup.+) in mol/L; (% G/C) is the number of G and C residues as a
percentage of total residues in the duplex; (% F) is the percent
formamide in solution (wt/vol); and L is the number of nucleotides
in each strand of the duplex.
I. Production of ASA Intermediates
The present invention also provides methods for the production of
ascorbic acid intermediates in host cells. The present invention
encompasses methods wherein the levels of an enzymatic activity
couple the catabolic and productive pathways, e.g., those which
phosphorylate D-glucose at its 6th carbon and/or which
phosphorylates D-gluconate at its 6th carbon are decreased during
part or all of the culturing. The present invention encompasses
methods wherein the levels of an enzymatic activity which
phosphorylates D-glucose at its 6th carbon and/or the levels of an
enzymatic activity which phosphorylates D-gluconate at its 6th
carbon are increased during part or all of the culturing. The
present invention also encompasses a method wherein the levels of
an enzymatic activity which phosphorylates D-glucose as its 6th
carbon and/or the levels of an enzymatic activity which
phosphorylates D-gluconate at its 6th carbon are not modified or
are increased at the beginning of the culturing to facilitate
growth, that is, to produce cell biomass, and decreased during the
later phases of culturing to facilitate desired product
accumulation.
The ASA intermediate may be further converted to a desired end
product such as ASA or erythorbate. For the production of ASA
intermediates, any host cell which is capable of converting a
carbon source to DKG can be used. Preferred strains of the family
Enterobacteriaceae are those that produce 2,5-diketo-D-gluconic
acid from D-glucose solutions, including Pantoea, are described in
Kageyama et al. (1992) International Journal of Systematic
Bacteriology vol. 42, p. 203-210. In a preferred embodiment, the
host cell is Pantoea citrea having a deletion of part or all of a
polynucleotide that encodes an endogenous glucokinase (encoded by
nucleic acid as depicted in SEQ ID NO:1) and a deletion of part or
all of a polynucleotide that encodes an endogenous gluconokinase
(encoded by nucleic acid as depicted in SEQ ID NO:3).
The production of ASA intermediates can proceed in a fermentative
environment, that is, in an in vivo environment, or a
non-fermentative environment, that is, in an in vitro environment;
or combined in vivolin vitro environments. In the methods which are
further described infra, the host cell or the in vitro environment
further comprise a heterologous DKG reductase which catalyses the
conversion of DKG to KLG.
A. In vivo Biocatalytic Environment
The present invention encompasses the use of host cells comprising
a modification in a polynucleotide encoding an endogenous enzymatic
activity that phosphorylates D-glucose at its 6th carbon and/or a
modification in a polynucleotide encoding an enzymatic activity
that phosphorylates D-gluconate at its 6th carbon in the in vivo
production of ASA intermediates. Biocatalysis begins with culturing
the host cell in an environment with a suitable carbon source
ordinarily used by Enterobacteriaceae strains, such as a 6 carbon
sugar, for example, glucose, or a 6 carbon sugar acid, or
combinations of 6 carbon sugars and/or 6 carbon sugar acids. Other
carbon sources include, but are not limited to galactose, lactose,
fructose, or the enzymatic derivatives of such. In addition to an
appropriate carbon source, fermentation media must contain suitable
minerals, salts, cofactors, buffers and other components, known to
those of skill in the art for the growth of cultures and promotion
of the enzymatic pathway necessary for production of desired
end-products.
In one illustrative in vivo Pantoea pathway, D-glucose undergoes a
series of membrane productive steps through enzymatic conversions,
which may include the enzymes D-glucose dehydrogenase, D-gluconate
dehydrogenase and 2-keto-D-gluconate dehydrogenase to give
intermediates which may include, but are not limited to GA, KDG,
and DKG, see FIG. 1. These intermediates undergo a series of
intracellular reducing steps through enzymatic conversions, which
may include the enzymes 2,5-diketo-D-gluconate reductase (DKGR),
2-keto reductase (2-KR) and 5-keto reductase (5-KR) to give desired
end products which include but are not limited to KLG and IA. In a
preferred embodiment of the in vivo environment for the production
of ASA intermediates, 5-KR activity is deleted in order to prevent
the consumption of IA
If KLG is a desired intermediate, nucleic acid encoding DKGR is
recombinantly introduced into the Pantoea fermentation strain. Many
species have been found to contain DKGR particularly members of the
Coryneform group, including the genera Corynebacterium,
Brevibacterium, and Arthrobacter.
In some embodiments of the present invention, 2,5-DKGR from
Corynebacterium sp. strain SHS752001 (Grindley et al., 1988,
Applied and Environmental Microbiology 54: 1770-1775) is
recombinantly introduced into a Pantoea strain. Production of
recombinant 2,5 DKG reductase by Erwinia herbicola is disclosed in
U.S. Pat. No. 5,008,193 to Anderson et al. Other sources of DKG
reductase are provided in Table I.
The fermentation may be performed in a batch process or in a
continuous process. In a batch process, regardless of what is
added, all of the broth is harvested at the same time. In a
continuous system, the broth is regularly removed for downstream
processing while fresh substrate is added. The intermediates
produced may be recovered from the fermentation broth by a variety
of methods including ion exchange resins, absorption or ion
retardation resins, activated carbon,
concentration-crystallization, passage through a membrane, etc.
B. In Vitro Biocatalytic Environment
The invention provides for the biocatalytic production of ASA
intermediates, e.g., KDG, DKG and KLG, from a carbon source in an
in vitro or non-fermentative environment, such as in a bioreactor.
The cells are first cultured for growth and for the
non-fermentative process the carbon source utilized for growth is
eliminated, the pH is maintained at between about pH 4 and about pH
9 and oxygen is present.
Depending upon the desired intermediate being produced, the process
may require the presence of enzymatic co-factor. In a preferred
embodiment disclosed herein, the enzymatic co-factor is
regenerated. In some embodiments, KDG is the desired ASA
intermediate-produced, the bioreactor is provided with viable or
non-viable Pantoea citrea host cells comprising a modification in a
polynucleotide encoding an endogenous enzymatic activity that
phosphorylates D-glucose at its 6th carbon and/or a modification in
a polynucleotide encoding an enzymatic activity that phosphorylates
D-gluconate at its 6th carbon. In this embodiment, the host cell
also has a mutation in a gene encoding 2-keto-D-gluconate
dehydrogenase activity. In this embodiment, the carbon source is
biocatalytically converted through two productive steps, to KDG. In
this embodiment, there is no need for co-factor regeneration.
When DKG is the desired ASA intermediate, the bioreactor is
provided with viable or non-viable Pantoea citrea host cells
comprising a modification in a polynucleotide encoding an
endogenous enzymatic activity that phosphorylates D-glucose at its
6th carbon and/or a modification in a polynucleotide encoding an
enzymatic activity that phosphorylates D-gluconate at its 6th
carbon and a carbon source which is biocatalytically converted
through three productive steps, to DKG. In this embodiment, there
is no need for co-factor regeneration.
When KLG is the desired ASA intermediate, the bioreactor is
provided with viable or non-viable Pantoea citrea host cells
comprising a modification in a polynucleotide encoding an
endogenous enzymatic activity that phosphorylates D-glucose at its
6th carbon and/or a modification in a polynucleotide encoding ah
enzymatic activity that phosphorylates D-gluconate at its 6th
carbon and a carbon source, such as D-glucose, which is
biocatalytically converted through three productive steps, and one
reducing step to KLG. In this embodiment, the reductase activity
may be encoded by nucleic acid contained within the Pantoea citrea
host cell or provided exogenously. In this embodiment, the first
productive enzymatic activity requires an oxidized form of the
co-factor and the reducing enzymatic activity requires a reduced
form of co-factor. In a preferred embodiment disclosed herein, the
Pantoea citrea cell is modified to eliminate the naturally
occurring GDH activity and a heterologous GDH activity, such as one
obtainable from T. acidophilum, Cryptococcus uniguttalatus or
Bacillus species and having a specificity for NADPH+, is introduced
into the Pantoea cell in order to provide a co-factor recycling
system which requires and regenerates one co-factor. In this
embodiment, the host cell further comprises nucleic acid encoding a
2,5-DKG reductase activity or the 2,5-DKG reductase is added
exogenously to the bioreactor.
In another embodiment for making KLG, the bioreactor is charged
with Pantoea citrea cells comprising a modification in nucleic acid
encoding an endogenous enzymatic activity which phosphorylates
D-glucose at its 6th carbon and/or in nucleic acid encoding an
enzymatic activity that phosphorylates D-gluconate at its 6th
carbon and further comprises nucleic acid encoding membrane-bound
GDH, appropriate enzymes and cofactor, and D-gluconic acid is added
which is converted to DKG. The reaction mixture is then made
anaerobic and glucose is added. The GDH converts the glucose to GA,
and the reductase converts DKG to KLG, while cofactor is recycled.
When these reactions are completed, oxygen is added to convert GA
to DKG, and the cycles continue.
In the in vitro biocatalytic process, the carbon source and
metabolites thereof proceed through enzymatic oxidation steps or
enzymatic oxidation and enzymatic reducing steps which may take
place outside of the host cell intracellular environment and which
exploit the enzymatic activity associated with the host cell and
proceed through a pathway to produce the desired ASA intermediate.
The enzymatic steps may proceed sequentially or simultaneously
within the bioreactor and some have a co-factor requirement in
order to produce the desired ASA intermediate. The present
invention encompasses an in vitro process wherein the host cells
are treated with an organic substance, such that the cells are
non-viable, yet enzymes remain available for oxidation and
reduction of the desired carbon source and/or metabolites thereof
in the biocatalysis of carbon source to ASA intermediate.
The bioreactor may be performed in a batch process or in a
continuous process. In a batch system, regardless of what is added,
all of the broth is harvested at the same time. In a continuous
system, the broth is regularly removed for downstream processing
while fresh substrate is added. The intermediates produced may be
recovered from the fermentation broth by a variety of methods
including ion exchange resins, absorption or ion retardation
resins, activated carbon, concentration-crystallization, passage
through a membrane, etc.
In some embodiments, the host cell is permeabilized or lyophilized
(Izumi et al., J. Ferment. Technol. 61 (1983) 135-142) as long as
the necessary enzymatic activities remain available to convert the
carbon source or derivatives thereof. The bioreactor may proceed
with some enzymatic activities being provided exogenously and in an
environment wherein solvents or long polymers are provided which
stabilize or increase the enzymatic activities. In some
embodiments, methanol or ethanol is used to increase reductase
activity. In another embodiment, Gafquat is used to stabilise the
reductase (see Gibson et al., U.S. Pat. No. 5,240,843).
In some embodiments of the invention, a carbon source is converted
to KLG in a process which involves co-factor regeneration. In this
enzymatic cofactor regeneration process, one equivalent of
D-glucose is oxidized to one equivalent of D-gluconate, and one
equivalent of NADP+ is reduced to one equivalent of NADPH by the
catalytic action of GDH. The one equivalent D-gluconate produced by
the GDH is then oxidized to one equivalent of 2-KDG, and then to
one equivalent of 2,5-DKG by the action of membrane bound
dehydrogenases GADH and KDGDH, respectively. The one equivalent
2,5-DKG produced is then reduced to one equivalent of 2-KLG, and
the NADPH is oxidized back to one equivalent of NADP+ by the action
of 2,5-DKG reductase, effectively recycling the equivalent cofactor
to be available for a second equivalent of D-glucose oxidation.
Other methods of cofactor regeneration can include chemical,
photochemical, and electrochemical means, where the equivalent
oxidized NADP+ is directly reduced to one equivalent of NADPH by
either chemical, photochemical, or electrochemical means.
C. Host Cells Producing ASA
Any productive or reducing enzymes necessary for directing a host
cell carbohydrate pathway into an ASA intermediate, such as, for
example, KDG, DKG or KLG, can be introduced via recombinant DNA
techniques known to those of skill in the art if such enzymes are
not naturally occurring in the host cell. Alternatively, enzymes
that would hinder a desired pathway can be inactivated by
recombinant DNA methods. The present invention encompasses the
recombinant introduction or inactivation of any enzyme or
intermediate necessary to achieve a desired pathway.
In some embodiments, Enterobacteriaceae strains that have been
cured of a cryptic plasmid are used in the production of ASA, see
PCT WO 98/59054.
In some embodiments, the host cell used for the production of an
ASA intermediate is Pantoea citrea, for example, ATCC accession
number 39140. Sources for nucleic acid encoding productive or
reducing enzymes which can be used in the production of ASA
intermediates in Pantoea species include the following:
TABLE-US-00001 TABLE I ENZYME CITATION glucose dehydrogenase Smith
et al. 1989, Biochem. J. 261: 973; Neijssel et al. 1989, Antonie
Van Leauvenhoek 56(1): 51-61 Cha, et al, Appl. Environ. Microbiol
63(1), 71-76 (1997); Pujol, C. J., et al, Microbiol. 145, 1217-1226
gluconic acid Matsushita et al. 1979, J. Biochem. dehydrogenase 85:
1173; Kulbe et al. 1987, Ann. N.Y. Acad Sci 6: 552 (Los Angeles)
Pujol, C. J., et at, J. of Bacteriol 63(1), 71-76 (1999) Yum, D, et
al, J. of Bacteriol 183(8)2230-2237 2-keto-D-gluconic acid
Stroshane 1977 Biotechnol. BioEng dehydrogenase 19(4) 459 2-keto
gluconate J. Gen. Microbiol. 1991, 137: 1479 reductase Pujols, et
al, J. of Bacterial. 182(8), (2000) 2,5-diketo-D-gluconic U.S. Pat.
Nos.: acid reductase 5,795,761; 5,376,544; 5,583,025; 4,757,012;
4,758,514; 5,008,193; 5,004,690; 5,032,514
D. Recovery of ASA Intermediates
Once produced, the ASA intermediates can be recovered and/or
purified by any means known to those of skill in the art,
including, lyophilization, crystallization, spray-drying, and
electrodialysis, etc. Electrodialysis methods for purifying ASA and
ASA intermediates such as KLG are described in for example, U.S.
Pat. No. 5,747,306 issued May 5, 1998 and U.S. Pat. No. 4,767,870,
issued Aug. 30, 1998. Alternatively, the intermediates can also be
formulated directly from the fermentation broth or bioreactor and
granulated or put in a liquid formulation.
KLG produced by a process of the present invention may be further
converted to ascorbic acid and the KDG to erythorbate by means
known to those of skill in the art, see for example, Reichstein and
Grussner, Helv. Chim. Acta., 17, 311-328 (1934). Four stereoisomers
of ascorbic acid are possible: L-ascorbic acid, D-araboascorbic
acid (erythorbic acid), which shows vitamin C activity,
L-araboascorbic acid, and D-xyloascorbic acid.
E. Assay Conditions
Methods for detection of ASA intermediates, ASA and ASA
stereoisomers include the use of redox-titration with 2,6
dichloroindophenol (Burton et al. 1979, J. Assoc. Pub. Analysts
17:105) or other suitable reagents; high-performance liquid
chromatography (HPLC) using anion exchange (J. Chrom. 1980,
196:163); and electro-redox procedures (Pachia, 1976, Anal. Chem.
48:364). The skilled artisan will be well aware of controls to be
applied in utilizing these detection methods.
Fermentation media in the present invention must contain suitable
carbon substrates which will include but are not limited to
monosaccharides such as glucose, oligosaccharides such as lactose
or sucrose, polysaccharides such as starch or cellulose and
unpurified mixtures from a renewable feedstocks such as cheese whey
permeate, cornsteep liquor, sugar beet molasses, and barley malt.
Additionally the carbon substrate may also be one-carbon substrates
such as carbon. While it is contemplated that the source of carbon
utilized in the present invention may encompass a wide variety of
carbon containing substrates and will only be limited by the choice
of organism, the preferred carbon substrates include glucose and/or
fructose and mixtures thereof. By using mixtures of glucose and
fructose in combination with the modified genomes described
elsewhere in this application, uncoupling of the oxidative pathways
from the catabolic pathways allows the use of glucose for improved
yield and conversion to the desired ascorbic acid intermediate
while utilizing the fructose to satisfy the metalbolic requirements
of the host cells.
Although it is contemplated that all of the above mentioned carbon
substrates are suitable in the present invention preferred are the
carbohydrates glucose, fructose or sucrose. The concentration of
the carbon substrate is from about 55% to about 75% on a
weight/weight basis.
Preferably, the concentration is from about 60 to about 70% on a
weight/weight basis. The inventors most preferably used 60% or 67%
glucose.
In addition to an appropriate carbon source, fermentation media
must contain suitable minerals, salts, vitamins, cofactors and
buffers suitable for the growth or the cultures and promotion of
the enzymatic pathway necessary for ascorbic acid intermediate
production.
Culture Conditions:
Precultures:
Typically cell cultures are grown at 25 to 32.degree. C., and
preferably about 28 or 29.degree. C. in appropriate media. While
the examples describe growth media used, other exemplary growth
media useful in the present invention are common commercially
prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose
(SD) broth or Yeast medium (YM) broth. Other defined or synthetic
growth media may also be used and the appropriate medium for growth
of the particular microorganism will be known by someone skilled in
the art of microbiology or fermentation science.
Suitable pH ranges preferred for the fermentation are between pH 5
to pH 8 where pH 7 to pH 7.5 for the seed flasks and between pH 5
to pH 6 for the reactor vessel.
It will be appreciated by one of skill in the art of fermentation
microbiology that, now that Applicants have demonstrated the
feasibility of the process of the present invention a number of
factors affecting the fermentation processes may have to be
optimized and controlled in order to maximize the ascorbic acid
intermediate production. Many of these factors such as pH, carbon
source concentration, and dissolved oxygen levels may affect the
enzymatic process depending on the cell types used for ascorbic
acid intermediate production.
Batch and Continuous Fermentations:
The present process employs a fed-batch method of fermentation for
its culture systems. A classical batch fermentation is a closed
system where the composition of the media is set at the beginning
of the fermentation and not subject to artificial alterations
during the fermentation. Thus, at the beginning of the fermentation
the media is inoculated with the desired organism or organisms and
fermentation is permitted to occur adding nothing to the system.
Typically, however, a "batch" fermentation is batch with respect to
the addition of carbon source and attempts are often made at
controlling factors such as pH and oxygen concentration. In batch
systems the metabolite and biomass compositions of the system
change constantly up to the time the fermentation is stopped.
Within batch cultures cells moderate through a static lag phase to
a high growth log phase and finally to a stationary phase where
growth rate is diminished or halted. If untreated, cells in the
stationary phase will eventually die. Cells in log phase generally
are responsible for the bulk of production of end product or
intermediate.
A variation on the standard batch system is the Fed-Batch system.
Fed-Batch fermentation processes are also suitable in the present
invention and comprise a typical batch system with the exception
that the substrate is added in increments as the fermentation
progresses. Fed-Batch systems are useful when catabolite repression
is apt to inhibit the metabolism of the cells and where it is
desirable to have limited amounts of substrate in the media.
Measurement of the actual substrate concentration in Fed-Batch
systems is difficult and is therefore estimated on the basis of the
changes of measurable factors such as pH, dissolved oxygen and the
partial pressure of waste gases such as CO.sub.2. Batch and
Fed-Batch fermentations are common and well known in the art and
examples may be found in Brock, supra.
Although the present invention is performed in batch mode it is
contemplated that the method would be adaptable to continuous
fermentation methods. Continuous fermentation is an open system
where a defined fermentation media is added continuously to a
bioreactor and an equal amount of conditioned media is removed
simultaneously for processing. Continuous fermentation generally
maintains the cultures at a constant high density where cells are
primarily in log phase growth.
Continuous fermentation allows for the modulation of one factor or
any number of factors that affect cell growth or end product
concentration. For example, one method will maintain a limiting
nutrient such as the carbon source or nitrogen level at a fixed
rate and allow all other parameters to moderate. In other systems a
number of factors affecting growth can be altered continuously
while the cell concentration, measured by media turbidity, is kept
constant. Continuous systems strive to maintain steady state growth
conditions and thus the cell loss due to media being drawn off must
be balanced against the cell growth rate in the fermentation.
Methods of modulating nutrients and growth factors for continuous
fermentation processes as well as techniques for maximizing the
rate of product formation are well known in the art of industrial
microbiology and a variety of methods are detailed by Brock,
supra.
It is contemplated that the present invention may be practiced
using either batch, fed-batch or continuous processes and that any
known mode of fermentation would be suitable. Additionally, it is
contemplated that cells may be immobilized on a substrate as whole
cell catalysts and subjected to fermentation conditions for
ascorbic acid intermediate production.
Identification and Purification of Ascorbic Acid Intermediates:
Methods for the purification of the desired ascorbic acid
intermediate from fermentation media are known in the art.
The specific ascorbic acid intermediate may be identified directly
by submitting the media to high pressure liquid chromatography
(HPLC) analysis. Preferred in the present invention is a method
where fermentation media is analyzed on an analytical ion exchange
column using a mobile phase of 0.01N sulfuric acid in an isocratic
fashion.
EXAMPLES
General Methods
Materials and Methods suitable for the maintenance and growth of
bacterial cultures were found in Manual of Methods for General
Bacteriology (Phillipp Gerhardt, R. G. E. Murray, Ralph N.
Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G.
Briggs Phillips, eds), pp. 210-213. American Society for
Microbiology, Washington, D.C. or Thomas D. Brock in Biotechnology:
A Textbook of Industrial Microbiology, Second Edition (1989)
Sinauer Associates, Inc., Sunderland, Mass. All reagents and
materials used for the growth, and of bacterial cells were obtained
from Diffco Laboratories (Detroit, Mich.), Aldrich Chemicals
(Milwaukee, Wis.) or Sigma Chemical Company (St. Louis, Mo.) unless
otherwise specified.
Growth medium for the precultures or inoculuum is commercially
available and preparations such as Luria Bertani (LB) broth,
Sabouraud Dextrose (SD) broth or Yeast medium (YM) broth are
obtainable from GIBCO/BRL (Gaithersburg, Md.). LB-50 amp is
Luria-Bertani broth containing 50 mu.g/ml ampicillin.
Fermentation Media:
Two basic fermentation media were prepared for use in the following
examples, and identified as Seed Flask Media and Fermentation
Media. These basic media were modified by altering the carbon
source or by the addition of other reagents such as sulfite. The
reagents useful for the respective media include KH.sub.2PO.sub.4,
K.sub.2HPO.sub.4, MgSO4.7H.sub.2O, Difco Soytone, Sodium citrate,
Fructose, (NH.sub.4).sub.2SO.sub.4, Nicotinic acid,
FeCl.sub.3.6H.sub.2O, and trace salts, including, but not limited
to ZnSO.sub.4.7H.sub.2O, MnSO.sub.4.H.sub.2O, and
Na.sub.2MoO.sub.4.2H.sub.2O); KH.sub.2PO.sub.4, MgSO4.7H2O,
(NH.sub.4).sub.2SO.sub.4, Mono-sodium glutamate,
ZnSO.sub.4.7H.sub.2O, MnSO.sub.4.H.sub.2O,
Na.sub.2MoO.sub.4.2H.sub.2O, FeCl.sub.3.6H.sub.2O, Choline
chloride, Mazu DF-204 (an antifoaming agent), Nicotinic acid,
Ca-pantothenate and HFCS (42DE). HFCS can also be made according to
the desired ratios of glucose to fructose, e.g., a frucose/glucose
solution made of 27.3 g/L powdered fructose, 25.0 g/L powdered
glucose.
Cells:
All commercially available cells used in the following examples
were obtained from the ATCC and are identified in the text by their
ATCC number. Recombinant P. citrea cells (ATCC39140) were used as
ascorbic acid intermediate producers and were constructed as
described in Examples 4 and 5. Enzymatic assays and genome analysis
revealed that the strains MDP41 and DD6 lacked the genes encoding
the glucokinase, gluconokinase and both enzymes whereas the
wild-type strains contained genes encoding the glucokinase and/or
gluconokinase enzymes.
Ascorbic Acid Intermediate Analysis:
The presence of ascorbic acid intermediates, e.g., 2-KLG, was
verified by running a HPLC analysis. Fermentation reactor vessel
samples were drawn off the tank and loaded onto Dionex (Sunnyvale,
Calif., Product No. 043118) Ion Pac AS 10 column (4 mm times 250
mm) connected to a Waters 2690 Separation Module and a Waters 410
Differential Refractometer (Milford, Mass.).
Methods of Assaying for Production of Ascorbic Acid
Intermediate
Methods for determining the yield, OUR, and CER were described
earlier in the definition section.
Recombinant Methods
Vector Sequences
Expression vectors used the methods of the present invention
comprise at least one promoter associated with the enzyme, which
promoter is functional in the host cell. In one embodiment of the
present invention, the promoter is the wild-type promoter for the
selected enzyme and in another embodiment of the present invention,
the promoter is heterologous to the enzyme, but still functional in
the host cell. In one embodiment of the present invention, nucleic
acid encoding the enzyme is stably integrated into the
microorganism genome.
In some embodiments, the expression vector contains a multiple
cloning site cassette which preferably comprises at least one
restriction endonuclease site unique to the vector, to facilitate
ease of nucleic acid manipulation. In a preferred embodiment, the
vector also comprises one or more selectable markers. As used
herein, the term selectable marker refers to a gene capable of
expression in the host microorganism which allows for ease of
selection of those hosts containing the vector. Examples of such
selectable markers include but are not limited to antibiotics, such
as, erythromycin, actinomycin, chloramphenicol and
tetracycline.
A preferred plasmid for the recombinant introduction of
non-naturally occurring enzymes or intermediates into a strain of
Enterobacteriaceae is RSF1010, a mobilizable, but not self
transmissible plasmid which has the capability to replicate in a
broad range of bacterial hosts, including Gram- and Gram+ bacteria.
(Frey et al., 1989, The Molecular biology of IncQ plasmids. In:
Thomas (Ed.), Promiscuous Plasmids of Gram Negative Bacteria.
Academic Press, London, pp. 79-94). Frey et al. (1992, Gene
113:101-106) report on three regions found to affect the
mobilization properties of RSF1010.
Transformation
General transformation procedures are taught in Current Protocols
In Molecular Biology (vol. 1, edited by Ausubel et al., John Wiley
& Sons, Inc. 1987, Chapter 9) and include calcium phosphate
methods, transformation using DEAE-Dextran and electroporation. A
variety of transformation procedures are known by those of skill in
the art for introducing nucleic acid encoding a desired protein in
a given host cell. A variety of host cells can be used for
recombinantly producing the pathway enzymes to be added
exogenously, including bacterial, fungal, mammalian, insect and
plant cells.
In some embodiments of the process, the host cell is an
Enterobacteriaceae. Included in the group of Enterobacteriaceae are
Erwinia, Enterobacter, Gluconobacter and Pantoea species. In the
present invention, a preferred Enterobacteriaceae fermentation
strain for the production of ASA intermediates is a Pantoea species
and in particular, Pantoea citrea. In some embodiments, the host
cell is Pantoea citrea comprising pathway enzymes capable of
converting a carbon source to KLG.
Identification of Transformants
Whether a host cell has been transformed can be detected by the
presence/absence of marker gene expression which can suggest
whether the nucleic acid of interest is present However, its
expression should be confirmed. For example, if the nucleic acid
encoding a pathway enzyme is inserted within a marker gene
sequence, recombinant cells containing the insert can be identified
by the absence of marker gene function. Alternatively, a marker
gene can be placed in tandem with nucleic acid encoding the pathway
enzyme under the control of a single promoter. Expression of the
marker gene in response to induction or selection usually indicates
expression of the enzyme as well.
Alternatively, host cells which contain the coding sequence for a
pathway enzyme and express the enzyme may be identified by a
variety of procedures known to those of skill in the art. These
procedures include, but are not limited to, DNA-DNA or DNA-RNA
hybridization and protein bioassay or immunoassay techniques which
include membrane-based, solution-based, or chip-based technologies
for the detection and/or quantification of the nucleic acid or
protein.
Additionally, the presence of the enzyme polynucleotide sequence in
a host microorganism can be detected by DNA-DNA or DNA-RNA
hybridization or amplification using probes, portions or fragments
of the enzyme polynucleotide sequences.
The manner and method of carrying out the present invention may be
more fully understood by those of skill in the art by reference to
the following examples, which examples are not intended in any
manner to limit the scope of the present invention or of the claims
directed thereto. All references and patent publications referred
to herein are hereby incorporated by reference.
EXAMPLES
Example 1
Construction of a Genomic Library from P. citrea 139-2a
P. citrea genomic DNA was prepared using the DNA-Pure.TM. genomic
DNA Isolation Kit (CPG, Lincoln Park, N.J.). 50 micrograms of this
DNA was partially digested with the restriction enzyme Sau3A
accordingly the manufacturer recommendations (Roche Molecular
Biochemicals, Indianapolis, Ind.). The products of the digestion
were separated on a 1% agarose gel and the DNA fragments of 3-5
kilobases were purified from the gel using the Qiaquick Gel
extraction kit (Qiagen Inc. Valencia, Calif.). The resulting DNA
was ligated with BamH1H-linearized plasmid pBK-CMV (Stratagene, La
Jolla, Calif.). A library of around 10.times..times. different
plasmids was obtained in this way.
Example 2
Isolation of the Structural Gene for the Glucokinase Enzyme
To select for a plasmid that carries the glucokinase gene from P.
citrea, the genomic library (see above) was transformed into a E.
coli strain devoid of the glucokinase gene (glkA) and the PTS
transport system, strain NF9, glk.sup.-, (Flores et al., Nat.
Biotech. 14, 620-623). After transformation, the cells were
selected for growth on M9 media with glucose as the only carbon
source. With this strategy, plasmids able to complement the
glk.sup.- or pts.sup.- mutations were selected.
After 48 hrs. of incubation at 37.degree. C., many colonies were
visible. Several of these colonies were further purified and their
plasmids isolated and characterized by restriction analysis. It was
found that all the plasmids contained a common DNA fragment.
After re-transforming these plasmids back into NF9, glk.sup.-, all
of them allowed growth on M9-glucose media, corroborating that they
were able to complement at least one of the mutations present in
NF9, glk.sup.-.
Plasmid pMD4 was isolated in this way and contains an insert of
around 3.9 kb. The insert in this plasmid was sequenced and it was
found that in a region of around 1010 bp, a gene with a strong
similarity to the E. coli glkA gene was present. (SEQ ID 4.)
Example 3
Inactivation of the Glucokinase Gene by Homologous
Recombination
The general strategy to inactivate genes by homologous
recombination with a a suicide vector has been delineated before
(Miller and Mekalanos, J. Bacteriol. 170 (1988) 2575-2583). To
inactivate the glk gene from P. citrea by this approach two
plasmids were constructed: pMD5 and pMD6.
To construct pMD5, plasmid pMD4 was digested with the NcoI and
SnaB1 restriction enzymes accordingly manufacturer specifications.
(Roche Molecular Biochemicals, Indianapolis, Ind.). The cohesive
ends generated by these enzymes were blunt-ended with T4 polymerase
using standard techniques. This DNA was ligated with a
loxP-Cat-loxP cassette isolated from pLoxCat2 as a SpeI-EcoRV DNA
fragment. (Palmeros et al., Gene (2000) 247, 255-264.). This
cassette codes for Chloramphenicol resistance. The ligation mixture
was transformed into TOP10 competent cell (Invitrogen, Carlsbard
Calif.). selecting for growth on Chloramphenicol 10 micrograms/ml.
Several colonies were obtained after 18 hr. incubation at
37.degree. C. The plasmids of some of these colonies were purified
and characterized by restriction analysis. The presence of the
loxP-Cat-loxP and the deletion of the DNA region between the NcoI
and SnaB1 sites in the glk gene was confirmed. The plasmid with
these properties was named pMD5.
To construct pMD6, plasmid pMD5 was digested with the BamH1 and
Cel11 restriction enzymes. The DNA fragment containing the glk gene
interrupted with the loxP-cassette was ligated to a EcoRV-Bsa1 DNA
fragment isolated from plasmid pR6Kori1 (unpublished results). This
fragment contains the R6K origin of replication and the Kanamycin
resistance gene. The ligation mixture was transformed into strain
SY327 (Miller and Mekalanos., ibid.) and transformants were
selected on plates containing kanamycin and chloramphenicol (20 and
10 micrograms/ml respectively). Several colonies were obtained
after 24 hr. incubation at 37.degree. C. The plasmids of some of
these colonies were purified and characterized by restriction
analysis. The presence of the loxP-Cat-loxP and the R6K origin was
confirmed. The plasmid with these characteristics was named
pMD6.
One characteristic of pMD6 and R6K derivatives in general, is that
they can only replicate in strains that carry the pir gene from
plasmid R6K (Miller and Mekalanos., ibid.). P. citrea does not
contain the pir gene or sustains replication of pMD6. After
transforming pMD6 into P. citrea 139-2a and selecting for Cm (R)
strains, the proper gene replacement by homologous recombination
was obtained. The inactivation of the glucokinase gene was
confirmed by assaying Glucokinase activity using the
glucokinase-glucose-6-phosphate deydrogenase coupled assay
described by Fukuda et al., (Fukuda Y., Yamaguchi S., Shimosaka M.,
Murata K. and Kimura A. J. Bacteriol. (1983) vol. 156: pp.
922-925). The P. citrea strain where the glucokinase inactivation
was confirmed was named MDP4.
Further confirmation of the inactivation of the glucokinase gene
was generated by comparing the size PCR products obtained using
chromosomal DNA from 139-2a or MDP4 strains and primers that
hybridize with the glucokinase structural gene (SEQ. ID. 8, SEQ.
ID. 9). With this approach, the size of the PCR products should
reflect that the loxP-Cat-loxP cassette was cloned in the
glucokinase structural gene.
Example 4
Removal of the Chloramphenicol Resistance Marker in MDP4
After overnight growth on YENB medium (0.75% yeast extract, 0.8%
nutrient broth) at 30.degree. C., P. citrea MDP40 in a water
suspension was electrotransformed with plasmid pJW168 (. (Palmeros
et al., Gene (2000) 247, 255-264.). which contained the
bacteriophage P1 Cre recombinase gene (IPTG-inducible), a
temperature-sensitive pSC101 replicon, and an ampicillin resistance
gene. Upon outgrowth in SOC medium at 30.degree. C., transformants
were selected at 30.degree. C. (permissive temperature for pJW168
replication) on LB agar medium supplemented with carbenicillin (200
.mu.g/ml) and IPTG (1 mM). Two serial overnight transfers of pooled
colonies were carried out at 35.degree. C. on fresh LB agar medium
supplemented with carbenicillin and IPTG in order to allow excision
of the chromosomal chloramphenicol resistance gene via
recombination at the loxP sites mediated by the Cre recombinase
(Hoess and Abremski, J. Mol. Biol., 181:351-362). Resultant
colonies were replica-plated on to LB agar medium supplemented with
carbenicillin and IPTG and LB agar supplemented with
chloramphenicol (12.5 .mu.g/ml) to identify colonies at 30.degree.
C. that were carbenicillin-resistant and chloramphenicol-sensitive
indicating marker gene removal. An overnight 30.degree. C. culture
of one such colony was used to inoculate 10 ml of LB medium. Upon
growth at 30.degree. C. to OD (600 nm) of 0.6, the culture was
incubated at 35.degree. C. overnight. Several dilutions were plated
on prewarmed LB agar medium and the plates incubated overnight at
35.degree. C. (the non-permissive temperature for pJW168
replication). Resultant colonies were replica-plated on to LB agar
medium and LB agar medium supplemented with carbenicillin (200
.mu.g/ml) to identify colonies at 30.degree. C. that were
carbenicillin-sensitive, indicating loss of plasmid pJW168. One
such glK mutant, MDP41, was further analyzed by genomic PCR using
primers SEQ ID NO:5 and SEQ ID NO:6 and yielded the expected PCR
product (data not shown).
Example 5
Inactivation of the Gluconate Kinase Gene by Homologous
Recombination
The general strategy utilized to inactivate the gluconate kinase
gene of P. citrea is presented in FIG. 9, was in essence the same
used to inactivate the glucokinase gene as described in example 3.
Briefly, after isolating and sequencing a plasmid that allowed a E.
coli strain gntk idnK, to grow using gluconate as the only carbon
source (data not shown); a DNA fragment containing the structural
gene for the gluconate kinase gene was generated by PCR using
primers SEQ. ID NO: 10 and SEQ. ID NO:11. This approximately 3 kb
PCR product was cloned in a multicopy plasmid containing an R6K
origin of replication. A unique PstI restriction site located in
the gluconate kinase structural gene as shown in SEQ. ID NO: 2, was
utilized to insert a loxP-Cat-loxP cassette. This construction was
transferred to the chromosome of the P. citrea strain MDP41 by
homologous recombination.
The correct interruption of the gluconate kinase with the
loxP-Cat-loxP cassette was confirmed by PCR, using primers SEQ ID
NO:11 and SEQ ID NO:12.
The new strain, with both glucose and gluconate kinase inactivated
was named MDP5. This strain still contains the Cat marker inserted
in the gluconate kinase structural gene. By repeating the procedure
described in example 4, a marker-less strain was obtained and named
DD6.
Experimental 6
The following illustrates the benefit of a double delete host cell
(glucokinase and gluconokinase deleted Pantoea host cells) in terms
of O.sub.2 demand.
Seed Train:
A vial of culture stored in liquid nitrogen is thawed in air and
0.75 mL is added to a sterile 2-L Erlenmeyer flasks containing 500
mL of seed medium. Flasks are incubated at 29.degree. C. and 250
rpm for 12 hours. Transfer criteria is an OD.sub.550 greater than
2.5.
Seed Flask Medium
A medium composition was made according to the following:
TABLE-US-00002 Component Amount KH.sub.2PO.sub.4 12.0 g/L
K.sub.2HPO.sub.4 4.0 g/L MgSO4.cndot.7H.sub.2O 2.0 g/L Difco
Soytone 2.0 g/L Sodium citrate 0.1 g/L Fructose 5.0 g/L
(NH.sub.4).sub.2SO.sub.4 1.0 g/L Nicotinic acid 0.02 g/L
FeCl.sub.3.cndot.6H.sub.2O 5 mL/L (of a 0.4 g/L stock solution)
Trace salts 5 mL/L (of the following solution: 0.58 g/L
ZnSO.sub.4.cndot.7H.sub.2O, 0.34 g/L MnSO.sub.4.cndot.H.sub.2O,
0.48 g/L Na.sub.2MoO.sub.4.cndot.2H.sub.2O)
The pH of the medium solution was adjusted to 7.0.+-.0.1 unit with
20% NaOH. Tetracycline HCl was added to a final concentration of 20
mg/L (2 mL/L of a 10 g/L stock solution). The resulting medium
solution was then filter sterilized with a 0.2.mu. filter unit. The
medium was then autoclaved and 500 mL of the previously autoclaved
medium was added to 2-L Erlenmeyer flasks.
Production Fermentor
Additions to the reactor vessel prior to sterilization
TABLE-US-00003 Component Conc KH.sub.2PO.sub.4 3.5 g/L
MgSO4.cndot.7H2O 1.0 g/L (NH.sub.4).sub.2SO.sub.4 0.92 g/L
Mono-sodium glutamate 15.0 g/L ZnSO.sub.4.cndot.7H.sub.2O 5.79 mg/L
MnSO.sub.4.cndot.H.sub.2O 3.44 mg/L
Na.sub.2MoO.sub.4.cndot.2H.sub.2O 4.70 mg/L
FeCl.sub.3.cndot.6H.sub.2O 2.20 mg/L Choline chloride 0.112 g/L
Mazu DF-204 0.167 g/L
The above constituted media was sterilized at 121.degree. C. for 45
minutes.
After tank sterilization, the following additions were made to the
fermentation tank:
TABLE-US-00004 Component Conc Nicotinic acid 16.8 mg/L
Ca-pantothenate 3.36 mg/L HFCS (42DE) 95.5 g/L (gluconate or
glucose if desired as the particular starting substrate)
The final volume after sterilization and addition of
post-sterilization components was 6.0 L. The so prepared tank and
medium were inoculated with the full entire contents from seed
flask prepared as described to give a volume of 6.5 L.
Growth conditions were at 29.degree. C. and pH 6.0. Agitation rate,
back pressure, and air flow are adjusted as needed to keep
dissolved oxygen above zero.
Results
The oxidative pathway for ascorbic acid intermediates is depicted
in FIG. 10. By determining the amount of carbon dioxide produce
(CER), one can calculate the amount of carbon utilized by the
catabolic pathway and thus measure the uncoupling of the catabolic
and productive (oxidative) pathways since the sole source of carbon
for CO.sub.2 is from the carbon substrate, no additional CO.sub.2
having been supplied into the reactor vessel. When the wild-type
organism was utilized in the fermentation process, 63% of the
glucose was converted to an ascorbic acid intermediate, while 37%
was converted, as measured by the CER, to catabolic products (FIG.
12). In the second phase of the study, the nucleic acid encoding
glucokinase expression was run under conditions of the wild-type.
As shown in FIG. 14, CO.sub.2 evolution decreased to about 18%, as
measured by CER. Thus glucose catabolism was reduced, but not
completely uncoupled. In an attempt to ascertain the source, i.e.
the pathway wherein the carbon substrate was being diverted to the
catabolic pathway, gluconic acid was provided as the sole carbon
source. As shown in FIG. 15 in comparison with FIG. 14, gluconic
acid was catabolized at about the same rate as if glucose had been
the carbon substrate. (83% gluconate converted to ascorbic acid
intermediate v. 17% of the gluconic acid converted to the catabolic
pathway (as measured by CER). See also Table 2:
TABLE-US-00005 TABLE 2 Fraction of Fraction of Glucose Gluconate
converted to converted to strain Metabolism DKG Metabolism DKG
Wild-type 0.37 0.63 -- -- Glucokinase 0.18 0.82 0.17 0.83 delete
(glkA) Gluconokinase 0.24 0.76 0.02 0.98 delete (gntK)
A last phase of the study was provided by the examination of the
OUR and CER of a host cell having the genomic encoding for
glucokinase and gluconokinase deleted from the host cell genome.
FIG. 16 depicts 3% glucose was converted to CO.sub.2, whereas a
control (wild-type) exhibited a 43% glucose to CO.sub.2 yield. As a
result, it appears that the wild-type exhibited a high catabolism
of glucose by the catabolic pathway, which resulted in reduced
yield and a high oxygen requirement. However, a dual deletion of
glucokinase and gluconokinase essentially inactivated catabolism to
less than 10 percent, less than 5 percent and particularly 3 or
less % of the initial carbon substrate.
Conclusions
The double mutant of glucokinase and gluconokinase appeared to
shunt almost all of the glucose to 2,5-DKG, about 98%.
Example 7
Production of Glycerol from Fructose
To demonstrate that Pantoea citrea can be used to produce chemical
compounds derived from fructose, glycerol was produced using the
approach described by Empatage et al., [Emptage, M., Haynie, S.,
Laffend, L., Pucci, J. and Whited, G. Process for the biological
production of 1,3-propanediol with high titer. Patent: WO 0112833-A
41 22, Feb., 2001; E.I. DU PONT DE NEMOURS AND COMPANY; GENENCOR
INTERNATIONAL, INC.]. Briefly, this approach uses two enzymes from
yeast to convert dihydroxyacetone phosphate (DHAP) into glycerol as
shown in the following reaction:
The genes for the GPD1 and GPP2 enzymes were cloned in a multicopy
plasmid pTrc99 under the control of the Trc promoter (Empatage et
al., 2001). This plasmid (pAH48) is able to produce high levels of
both enzymes. The inventors recognized that to produce glycerol in
P. citrea, it was desireable to eliminate or reduce the natural
ability of the strain to assimilate glycerol. A common glycerol
catabolic pathway in many bacteria, is through the action of the
glycerol kinase [Lin E. C. Ann. Rev. Microbiol. 1976. 30:535-578.
Glycerol dissimilation and its regulation in bacteria]. The
inventors found that the P. citrea was able to grow in media
containing glycerols as the only carbon source. Furthermore,
inspection of the P. citrea genome sequence, showed that it
possesses a glycerol kinase gene, very similar to the glkA gene
from E. coli.
Thus, to eliminate the glycerol kinase activity, the structural
gene of this enzyme (gene glpK) was inactivated. This was
accomplished as described in Examples 2 and 5 (inactivation of
glucokinase and gluconokinase genes). Briefly, a 2.9 kb DNA
fragment containing the glpK gene and flanking sequences, was
obtained by PCR using chromosomal DNA from P. citrea and the
primers disclosed in SEQ ID NO: 11 and SEQ ID NO: 12. This 2.9 kb
DNA fragment was cloned in a R6K vector indicated in Examples 3 and
5. The DNA sequence of the glpK gene is shown in SLO ID NO: 13 and
the protein sequence of GlpK is shown in SEQ ID NO:14.
Inspection of the glpK DNA sequence showed the presence of a Hpa1
site, which was chosen to insert the LoxP-Cat-LoxP cassette. Once
the desired plasmid construction was obtained, the glpK
interruption was transferred to the chromosome of P. citrea strain
139-2a ps-, by homologous recombination as described in example 3
and 5. The resulting P. citrea glpK::Cm strain was named MDG1.
Once the interruption of the glpK gene in the P. citrea genome was
confirmed, the effect of this mutation was evaluated. For such a
purpose, strain MDG1 was grown in minimal media M9 containing
glycerol 0.4% as the only carbon source. After incubating the cells
for 48 hours at 30.degree. C., no growth was observed, indicating
that strain MDG1 lost the ability to utilize glycerol as a carbon
source.
Strain MDG1 was transformed with plasmid pAH48 (Emptage et al.,
2001), and the resulting strain MDG2, was tested for its capacity
to produce glycerol using fructose as the only carbon source. This
was accomplished by incubating the strain in minimal media
containing 2% fructose as the only carbon source. After incubating
the cells for 24 hours at 30.degree. C., a sample was collected and
analyzed by HPLC as described by Emptage et al. (2001). By doing
this, it was found that strain MDG1 did not produce any glycerol,
while strain MDG2 produced 1.36 g/L of glycerol. These results
demonstrated that P. citrea was able to divert a substantial part
of fructose into the formation of glycerol.
Various other examples and modifications of the foregoing
description and examples will be apparent to a person skilled in
the art after reading the disclosure without departing from the
spirit and scope of the invention, and it is intended that all such
examples or modifications be included within the scope of the
appended claims. All publications and patents referenced herein are
hereby incorporated by reference in their entirety.
SEQUENCE LISTINGS
1
14 1 963 DNA Pantoea citrea 1 atgacaaact atgccttggt cggcgatgta
ggcggaacta acgcccgcct tgcgttgtgt 60 gatgtgactg acggcagcat
ctcgcaggcc aaaacctttt caaccgagga ttaccagagc 120 ctggaagatg
ttattcgtga gtatctggcg gatcaacaag ccatcacctg tgcatctatc 180
gccatcgcct gtccggtgaa agatgactgg attgaaatga ctaatcatag ctgggcgttc
240 tctatcagtg agatgaaaca aaatctcggg ctggaacatc tggaagtgat
taacgatttc 300 actgcggtct ccatggcaat tccaatgctg ggcagtgacg
atgtcattca gttcggcggt 360 ggtgcaccgg taaaagataa accgatagct
atctatggtg ccggaacagg actgggggtg 420 agccatctgg ttcatgtcaa
caaacactgg gtcagcttgc ctggtgaagg cggacatgta 480 gatttcacct
gtggtaccga agaagaagac atgatcatga gtgtgctgcg tgcagaacgt 540
ggccgggtgt cagctgaacg ggtgctgtca ggaaaaggtc tggtgaatat ttaccgggcc
600 attgtgattt ctgacaaccg tgttcctgaa cgtctgcaac ctcaggacgt
aaccgagcgt 660 gcattatccg gaagctgtac tgactgtcgt cgtgcactgt
cattgttctg tgtgattatg 720 ggacgttttg gcgggaacct ggccctgaca
cttggaacct tcggtggggt gtatattgcc 780 ggcggaattg ttccacgctt
cctgcagttc tttaaagcct ccggtttccg tgctgctttc 840 gaagataagg
gacgtttccg ttcttacgta caggatattc cggtctatct gattacccat 900
gatcagccgg ggctgctggg tgccggtgcc catatgcgcc agactttagg gatggaactg
960 taa 963 2 320 PRT Pantoea citrea 2 Met Thr Asn Tyr Ala Leu Val
Gly Asp Val Gly Gly Thr Asn Ala Arg 1 5 10 15 Leu Ala Leu Cys Asp
Val Thr Asp Gly Ser Ile Ser Gln Ala Lys Thr 20 25 30 Phe Ser Thr
Glu Asp Tyr Gln Ser Leu Glu Asp Val Ile Arg Glu Tyr 35 40 45 Leu
Ala Asp Gln Gln Ala Ile Thr Cys Ala Ser Ile Ala Ile Ala Cys 50 55
60 Pro Val Lys Asp Asp Trp Ile Glu Met Thr Asn His Ser Trp Ala Phe
65 70 75 80 Ser Ile Ser Glu Met Lys Gln Asn Leu Gly Leu Glu His Leu
Glu Val 85 90 95 Ile Asn Asp Phe Thr Ala Val Ser Met Ala Ile Pro
Met Leu Gly Ser 100 105 110 Asp Asp Val Ile Gln Phe Gly Gly Gly Ala
Pro Val Lys Asp Lys Pro 115 120 125 Ile Ala Ile Tyr Gly Ala Gly Thr
Gly Leu Gly Val Ser His Leu Val 130 135 140 His Val Asn Lys His Trp
Val Ser Leu Pro Gly Glu Gly Gly His Val 145 150 155 160 Asp Phe Thr
Cys Gly Thr Glu Glu Glu Asp Met Ile Met Ser Val Leu 165 170 175 Arg
Ala Glu Arg Gly Arg Val Ser Ala Glu Arg Val Leu Ser Gly Lys 180 185
190 Gly Leu Val Asn Ile Tyr Arg Ala Ile Val Ile Ser Asp Asn Arg Val
195 200 205 Pro Glu Arg Leu Gln Pro Gln Asp Val Thr Glu Arg Ala Leu
Ser Gly 210 215 220 Ser Cys Thr Asp Cys Arg Arg Ala Leu Ser Leu Phe
Cys Val Ile Met 225 230 235 240 Gly Arg Phe Gly Gly Asn Leu Ala Leu
Thr Leu Gly Thr Phe Gly Gly 245 250 255 Val Tyr Ile Ala Gly Gly Ile
Val Pro Arg Phe Leu Gln Phe Phe Lys 260 265 270 Ala Ser Gly Phe Arg
Ala Ala Phe Glu Asp Lys Gly Arg Phe Arg Ser 275 280 285 Tyr Val Gln
Asp Ile Pro Val Tyr Leu Ile Thr His Asp Gln Pro Gly 290 295 300 Leu
Leu Gly Ala Gly Ala His Met Arg Gln Thr Leu Gly Met Glu Leu 305 310
315 320 3 531 DNA Pantoea citrea 3 atgagtacag cttcttcaaa tcatcatgtg
tttatcctga tgggcgtttc cggcagcgga 60 aagtcggtgg tcgccaatcg
tgtctcttac cagttgcaaa ccgcatttct tgatggtgac 120 tttctgcatc
ccagagcgaa catcatgaaa atggctgacg ggcatccgct caatgatcag 180
gatcgtcaac cctggctgca ggccattaat gatgcggctt ttgctatgca gcggacccag
240 gctgtatcgt taattgtgtg ttcgtcactg aaaaaaagtt atcgcgatat
tcttcgtgaa 300 ggtaacagca atcttaagtt tgtttatctg aaaggtgact
tcgataccat cgaatcgcgt 360 cttaaagccc gcaagggaca cttcttcaaa
cccgccatgc tggtaacaca attcgcaact 420 ctcgaagagc cgaccccgga
tgaaactgat gtcctcacgg tggatatccg gcagtcgctg 480 gatgaggttg
ttgctgccac ggtagaagcg atcaaacacg caattcagta a 531 4 176 PRT Pantoea
citrea 4 Met Ser Thr Ala Ser Ser Asn His His Val Phe Ile Leu Met
Gly Val 1 5 10 15 Ser Gly Ser Gly Lys Ser Val Val Ala Asn Arg Val
Ser Tyr Gln Leu 20 25 30 Gln Thr Ala Phe Leu Asp Gly Asp Phe Leu
His Pro Arg Ala Asn Ile 35 40 45 Met Lys Met Ala Asp Gly His Pro
Leu Asn Asp Gln Asp Arg Gln Pro 50 55 60 Trp Leu Gln Ala Ile Asn
Asp Ala Ala Phe Ala Met Gln Arg Thr Gln 65 70 75 80 Ala Val Ser Leu
Ile Val Cys Ser Ser Leu Lys Lys Ser Tyr Arg Asp 85 90 95 Ile Leu
Arg Glu Gly Asn Ser Asn Leu Lys Phe Val Tyr Leu Lys Gly 100 105 110
Asp Phe Asp Thr Ile Glu Ser Arg Leu Lys Ala Arg Lys Gly His Phe 115
120 125 Phe Lys Pro Ala Met Leu Val Thr Gln Phe Ala Thr Leu Glu Glu
Pro 130 135 140 Thr Pro Asp Glu Thr Asp Val Leu Thr Val Asp Ile Arg
Gln Ser Leu 145 150 155 160 Asp Glu Val Val Ala Ala Thr Val Glu Ala
Ile Lys His Ala Ile Gln 165 170 175 5 24 DNA Artificial Sequence
primer 5 ttttcaaccg aggattacca gagc 24 6 24 DNA Artificial Sequence
primer 6 cacggcgcag gaatgataca gaga 24 7 23 DNA Artificial Sequence
primer 7 gggaaggttc tgatgtgtcc gtg 23 8 22 DNA Artificial Sequence
primer 8 gccggttgca gcgcgtgacc gc 22 9 24 DNA Artificial Sequence
primer 9 actaaaaggg tacggtgtca gaga 24 10 24 DNA Artificial
Sequence primer 10 gtgttgcggt acttatcatt atta 24 11 21 DNA
Artificial Sequence primer 11 tgcagtttca atgggtgttt a 21 12 20 DNA
Artificial Sequence primer 12 tgtccggcat gcaggtcaga 20 13 1518 DNA
Pantoea citrea 13 atgactaacg ctgaaaacaa atacattgtt gcactggacc
agggaaccac cagctcacga 60 gcggtagtac tggatcacga tgcaaatatt
atcgcggttt cacaacgtga atttactcag 120 cactatccta aaacaggctg
ggttgagcat gacccgatgg atatctgggc aacccagagt 180 tcaactctgg
tagaagtact ggcacacgcc gatattcgtt ctgatcagat tgcggcgatt 240
ggtattacta accagcgtga aaccaccatc gtctgggata agaaaaccgg caagcctgtc
300 tataacgcaa ttgtctggca ggacccacgc accgctgact actgctcaaa
actgaaaaaa 360 gaaggtcttg aagaatatat tcagaagacg accgggcttg
tgattaaccc ttacttctcc 420 ggaaccaaaa taaaatggat tctggacaat
gtggaaggtg cccgggatcg agccaaacgt 480 ggggaactgt tatttggtac
cgttgacacc tggctggtct ggaaaatgac tcagggtcgt 540 gtgcatgtta
ccgactttac caatgcttca cgtaccatga tatttgatat tcacaatctg 600
aagtgggatg accgtatgct ggacatcctt gatattccac gtgaaatgct gccagaagtt
660 aaagcatctt ctgaagttta cgggcagaca aacatcggtg gtaaaggcgg
aacccgtatt 720 ccgatcgccg ggatcgctgg tgatcagcag gcggctttat
acggccagct ctgtgtgcaa 780 ccaggtatgg cgaagaatac gtatggtacc
ggctgcttta tgttaatgaa taccggtaca 840 gaagcagtag cttctactca
tggcctgctg acaacaattg cctgcggtcc acggggtgaa 900 gttaactatg
cgctggaagg tgcagtcttt attggcggtg cttccattca atggctgcgt 960
gatgagatga aactgttctc tgaagcttta gactctgaat atttcgccac caaagtaaaa
1020 gactctaacg gggtttatat ggtgccggca tttaccggtt taggcgctcc
gtactgggac 1080 ccatatgccc gtggagcaat ttttggcctg acccgcggaa
ccaatgctaa ccatattatc 1140 cgcgctactc tggaatctat tgcctaccag
actcgcgacg tgctggaagc aatgcagaat 1200 gatgcgaata cccgtctgca
gtcattgcgg gtagatggtg gcgctgtggc gaataatttc 1260 ctgatgcaat
tccagtccga tattctcgga acacgggttg agcgtccgga agttcgtgaa 1320
gtcaccgctc ttggagctgc ctatctggcc gggctggcag ttggattctg gaaagatctg
1380 gatgaagtcc gttcgaaagc ggttattgag cgcgagttcc gcccttcaat
cgaaacgact 1440 gaacgtaact tccgttatgc cggctggaaa aaagctgttt
cccgcgccct gcgctgggaa 1500 gatgaaaacg aacaataa 1518 14 505 PRT
Pantoea citrea 14 Met Thr Asn Ala Glu Asn Lys Tyr Ile Val Ala Leu
Asp Gln Gly Thr 1 5 10 15 Thr Ser Ser Arg Ala Val Val Leu Asp His
Asp Ala Asn Ile Ile Ala 20 25 30 Val Ser Gln Arg Glu Phe Thr Gln
His Tyr Pro Lys Thr Gly Trp Val 35 40 45 Glu His Asp Pro Met Asp
Ile Trp Ala Thr Gln Ser Ser Thr Leu Val 50 55 60 Glu Val Leu Ala
His Ala Asp Ile Arg Ser Asp Gln Ile Ala Ala Ile 65 70 75 80 Gly Ile
Thr Asn Gln Arg Glu Thr Thr Ile Val Trp Asp Lys Lys Thr 85 90 95
Gly Lys Pro Val Tyr Asn Ala Ile Val Trp Gln Asp Pro Arg Thr Ala 100
105 110 Asp Tyr Cys Ser Lys Leu Lys Lys Glu Gly Leu Glu Glu Tyr Ile
Gln 115 120 125 Lys Thr Thr Gly Leu Val Ile Asn Pro Tyr Phe Ser Gly
Thr Lys Ile 130 135 140 Lys Trp Ile Leu Asp Asn Val Glu Gly Ala Arg
Asp Arg Ala Lys Arg 145 150 155 160 Gly Glu Leu Leu Phe Gly Thr Val
Asp Thr Trp Leu Val Trp Lys Met 165 170 175 Thr Gln Gly Arg Val His
Val Thr Asp Phe Thr Asn Ala Ser Arg Thr 180 185 190 Met Ile Phe Asp
Ile His Asn Leu Lys Trp Asp Asp Arg Met Leu Asp 195 200 205 Ile Leu
Asp Ile Pro Arg Glu Met Leu Pro Glu Val Lys Ala Ser Ser 210 215 220
Glu Val Tyr Gly Gln Thr Asn Ile Gly Gly Lys Gly Gly Thr Arg Ile 225
230 235 240 Pro Ile Ala Gly Ile Ala Gly Asp Gln Gln Ala Ala Leu Tyr
Gly Gln 245 250 255 Leu Cys Val Gln Pro Gly Met Ala Lys Asn Thr Tyr
Gly Thr Gly Cys 260 265 270 Phe Met Leu Met Asn Thr Gly Thr Glu Ala
Val Ala Ser Thr His Gly 275 280 285 Leu Leu Thr Thr Ile Ala Cys Gly
Pro Arg Gly Glu Val Asn Tyr Ala 290 295 300 Leu Glu Gly Ala Val Phe
Ile Gly Gly Ala Ser Ile Gln Trp Leu Arg 305 310 315 320 Asp Glu Met
Lys Leu Phe Ser Glu Ala Leu Asp Ser Glu Tyr Phe Ala 325 330 335 Thr
Lys Val Lys Asp Ser Asn Gly Val Tyr Met Val Pro Ala Phe Thr 340 345
350 Gly Leu Gly Ala Pro Tyr Trp Asp Pro Tyr Ala Arg Gly Ala Ile Phe
355 360 365 Gly Leu Thr Arg Gly Thr Asn Ala Asn His Ile Ile Arg Ala
Thr Leu 370 375 380 Glu Ser Ile Ala Tyr Gln Thr Arg Asp Val Leu Glu
Ala Met Gln Asn 385 390 395 400 Asp Ala Asn Thr Arg Leu Gln Ser Leu
Arg Val Asp Gly Gly Ala Val 405 410 415 Ala Asn Asn Phe Leu Met Gln
Phe Gln Ser Asp Ile Leu Gly Thr Arg 420 425 430 Val Glu Arg Pro Glu
Val Arg Glu Val Thr Ala Leu Gly Ala Ala Tyr 435 440 445 Leu Ala Gly
Leu Ala Val Gly Phe Trp Lys Asp Leu Asp Glu Val Arg 450 455 460 Ser
Lys Ala Val Ile Glu Arg Glu Phe Arg Pro Ser Ile Glu Thr Thr 465 470
475 480 Glu Arg Asn Phe Arg Tyr Ala Gly Trp Lys Lys Ala Val Ser Arg
Ala 485 490 495 Leu Arg Trp Glu Asp Glu Asn Glu Gln 500 505
* * * * *